Anti-hepatitis b virus antibodies and use thereof

ABSTRACT

Antibodies (especially humanized antibodies) against the hepatitis B surface antigen (HBsAg), a nucleic acid molecule encoding same, a method for preparing same, and a pharmaceutical composition containing same. The anti-HBsAg antibodies have a higher binding affinity to HBsAg at a neutral pH than at an acidic pH, thereby significantly enhancing virus clearance efficiency and prolonging virus inhibition time. The antibodies and pharmaceutical composition may be used to prevent and/or treat HBV infections or diseases related to HBV infection (such as hepatitis B) for use in neutralizing the virulence of HBV in the body of a subject (such as a human) to reduce a serum level of HBV DNA and/or HBsAg in the body of the subject, or to activate a humoral immune response of a subject (such as a person infected with chronic HBV or a patient who has chronic hepatitis B) against HBV.

TECHNICAL FIELD

The present invention relates to the field of molecular virology andimmunology, especially the field of treatment of hepatitis B virus (HBV)infection. Specifically, the present invention relates to an antibodyagainst hepatitis B virus surface antigen (HBsAg) and a nucleic acidencoding the antibody, and a use thereof. The anti-HBsAg antibody of thepresent invention has a higher binding affinity for HBsAg at neutral pHthan at acidic pH. The novel antibody can be used for the preventionand/or treatment of an HBV infection or a disease associated with HBVinfection (for example, hepatitis B), for neutralizing a virulence ofHBV in a subject (for example, a human), or for reducing a serum levelof HBV DNA and/or HBsAg in a subject. Therefore, the present inventionfurther relates to a use of the antibody and variant thereof in themanufacture of a pharmaceutical composition for the prevention and/ortreatment of an HBV infection or a disease related to an HBV infection(for example, hepatitis B), for neutralizing a virulence of HBV in asubject (for example, a human), for reducing a serum level of HBV DNAand/or HBsAg in a subject, or for activating a humoral immune responseto HBV in a subject (for example, a person with chronic HBV infection ora patient with chronic hepatitis B).

BACKGROUND ART

Hepatitis B virus infection, especially chronic HBV infection, is one ofthe most important public health problems in the world (Dienstag J L.Hepatitis B virus infection. N Engl J Med 2008 Oct. 2;359(14):1486-1500). Chronic HBV infection can lead to a series of liverdiseases such as chronic hepatitis B (CHB), liver cirrhosis (LC) andprimary hepatocellular carcinoma (HCC) (Liaw Y F, Chu C M. Hepatitis Bvirus infection. Lancet 2009 Feb. 14; 373(9663): 582-592). According toreports, there are currently about 2 billion people in the world whohave been infected with HBV, there are now about 350 million personswith chronic hepatitis B virus infections, the risk of these infectedpersons eventually dying from HBV infection-associated liver diseasescan reach 15% to 25%, and more than one million people die from suchdiseases each year worldwide (Dienstag J L., ibid; and Liaw Y F, et al.,ibid).

The current treatment drugs for chronic HBV infection can be dividedinto interferons (IFNs) and nucleoside or nucleotide analogs (NAs)(Dienstag J L., ibid.; Kwon H, Lok A S. Hepatitis B therapy. Nat RevGastroenterol Hepatol 2011 May; 8(5): 275-284; and Liaw Y F et al.,ibid.). For HBV-infected patients (such as CHB patients), theabove-mentioned drugs alone or in combination can effectively inhibitviral replication in the body and greatly reduce HBV DNA levels; inparticular, after 52 weeks or more of such treatments, the response ratewhere the HBV DNA level in the body is below the lower limit ofdetection (virological response) can reach 40-80% (Kwon H et al.,ibid.). However, the treatment with the above-mentioned drugs alone orin combination cannot completely eliminate the HBV virus in the infectedpersons, and the response rate of HBsAg negative conversion or HBsAgseroconversion (a sign of complete HBV virus clearance in the infectedperson) caused thereby is usually less than 5% (Kwon H et al., ibid.).

The development of new drugs for the treatment of chronic HBV infectionbased on immunological means is one of the important research directionsin this field. Immunotherapy for chronic HBV infection is usuallycarried out in two ways: active immunotherapy (its corresponding drugforms including vaccines, etc.) and passive immunotherapy (itscorresponding drug forms including antibodies, etc.). Activeimmunotherapy refers to administration of a therapeutic vaccine(including protein vaccine, peptide vaccine, nucleic acid vaccine, etc.)in order to stimulate the body of chronic HBV infected person toactively produce a cellular immune response (CTL effect, etc.) or/andhumoral immune response against HBV (antibodies, etc.), so as to achievethe purpose of inhibiting or eliminating HBV. Currently, there is nodefinitely significant and effective active immunotherapy drug/vaccinethat can be used to treat chronic HBV infection. Passive immunotherapy(taking antibody as an example) refers to administration of an antibodywith therapeutic properties to a HBV infected person, and a therapeuticeffect can be achieved by the antibody-mediated virus neutralization toblock HBV from infecting newborn hepatocytes, or by theantibody-mediated immune clearance to remove viruses and infected livercells from the body. At present, the anti-HBs polyclonal antibodypurified from the serum/plasma of those who had a response to aprophylactic hepatitis B vaccine or those who have recovered from HBVinfection, namely high-potency hepatitis B immunoglobulin (HBIG), hasbeen widely used to block mother-to-child vertical transmission of HBV,prevent HBV reinfection after liver transplantation in patients withchronic HBV infection, and prevent people accidentally exposed to HBVfrom being infected. However, the direct application of HBIG in thetreatment of HBV-infected patients (for example, CHB patients) has noobvious effect, and it has many limitations such as fewer sources forhigh-potency plasma, high price, unstable nature, and potential safetyissues.

Therefore, it is urgent and necessary to develop innovative treatmentmethods and drugs for HBV infected persons that can more effectivelyremove HBV virus, especially HBsAg.

Contents of the Present Invention

The present inventors have previously developed an anti-HBsAg humanizedantibody with excellent properties, which can neutralize the virulenceof HBV in vivo and reduce the serum levels of HBV DNA and/or HBsAg. Onthe basis of the previous research, the present inventors have paid alot of creative work to conduct in-depth research and engineering of thehumanized antibody, thereby developing an anti-HBsAg antibody withpH-dependent antigen binding ability. The anti-HBsAg antibody of thepresent invention has a higher binding affinity for HBsAg at neutral pHthan at acidic pH, so that the reuse of antibody is realized, theantibody half-life is significantly extended, and the efficiency of HBVclearance is enhanced. Furthermore, the present inventors obtain ascavenger antibody and further extend the antibody half-life byintroducing a mutation into the Fc region of the above-mentionedantibody to enhance its affinity to hFcRn or mFcγRII under neutralcondition.

The antibody of the present invention is extremely advantageous, sinceit not only retains the activity of reducing the serum level of HBV DNAand/or HBsAg, but also has a longer time of antigen suppression, therebygreatly reducing the injection dosage and administration frequency oftreatment, and having significant clinical value.

Antibody of the Present Invention

Therefore, in one aspect, the present invention provides an antibody orantigen-binding fragment thereof capable of specifically binding toHBsAg, in which the antibody or antigen-binding fragment thereof bindsto HBsAg with higher affinity at neutral pH than at acidic pH.

In certain embodiments, the neutral pH is pH 6.7 to pH 7.5, such as pH7.4.

In certain embodiments, the acidic pH is pH 4.0 to pH 6.5, such as pH6.0.

In certain embodiments, a ratio of K_(D) of binding to HBsAg at anacidic pH (for example, pH 6.0) to K_(D) of binding to HBsAg at neutralpH (for example, pH 7.4) (i.e., value of K_(D) (acidic pH)/K_(D)(neutral pH)), of the antibody or antigen-binding fragment thereof, isgreater than 1, for example not less than 1.5, not less than 2, not lessthan 3, not less than 4, not less than 5, not less than 6, not less than7, not less than 8, not less than 9, not less than 10, not less than 15,not less than 20, not less than 30, not less than 40, not less than 50,not less than 60, not less than 70, not less than 80, not less than 90,not less than 100, not less than 300, not less than 500, not less than800, not less than 1000, not less than 2000, not less than 5000, or notless than 10,000. In some embodiments, the value of K_(D) (acidicpH)/K_(D) (neutral pH) is greater than 1 and not greater than 10000, forexample, not greater than 5000, not greater than 2000, not greater than1000, not Greater than 900, not greater than 800, not greater than 700,not greater than 600, not greater than 500, not greater than 400, notgreater than 300, not greater than 200, not greater than 100, notgreater than 90, not greater than 80, not greater than 70, not greaterthan 60, not greater than 50, not greater than 40, not greater than 30,not greater than 20, or not greater than 10. The K_(D) can be measuredby a technique known in the art, for example, by SPR technique (forexample, Biacore).

In some embodiments, a ratio of K_(D) of binding to HBsAg at pH 6.0 toK_(D) of binding to HBsAg at pH 7.4 of the antibody or antigen-bindingfragment thereof, is greater than 1, for example not less than 1.5, notless than 2. In certain embodiments, the K_(D) value of the antibody ofthe invention at neutral pH may be 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M,10⁻¹²M or less. In some embodiments, the K_(D) value of the antibody ofthe present invention at acidic pH may be 10⁻⁹M, 10⁻⁸M, 10⁻⁷M, 10⁻⁶M ormore.

In certain embodiments, a ratio of EC50 of binding to HBsAg at an acidicpH (for example, pH 6.0) to EC50 of binding to HBsAg at neutral pH (forexample, pH 7.4) (i.e., value of EC50 (acidic pH)/EC50 (neutral pH)), ofthe antibody or antigen-binding fragment thereof, is greater than 1, forexample not less than 1.5, not less than 2, not less than 3, not lessthan 4, not less than 5, not less than 6, not less than 7, not less than8, not less than 9, not less than 10, not less than 15, not less than20, not less than 30, not less than 40, not less than 50, not less than60, not less than 70, not less than 80, not less than 90, not less than100, not less than 300, not less than 500, not less than 800, not lessthan 1000, not less than 2000, not less than 5000, or not less than10,000. In some embodiments, the value of EC50 (acidic pH)/EC50 (neutralpH) is greater than 1 and not greater than 10000, for example, notgreater than 5000, not greater than 2000, not greater than 1000, notgreater than 900, and not greater than 800, not greater than 700, notgreater than 600, not greater than 500, not greater than 400, notgreater than 300, not greater than 200, not greater than 100, notgreater than 90, not greater than 80, not greater than 70, not greaterthan 60, not greater than 50, not greater than 40, not greater than 30,not greater than 20, or not greater than 10. In some embodiments, theEC50 is measured by ELISA method, for example, calculated by theregression analysis of a dose-response curve generated by the ELISAmethod.

In certain embodiments, a ratio of EC50 of binding to HBsAg at pH 6.0 toEC50 of binding to HBsAg at pH 7.4 of the antibody or antigen-bindingfragment thereof, is greater than 1, for example, not less than 1.5, ornot less than 2.

In certain embodiments, the antibody or antigen-binding fragment thereofof the present invention is derived from the anti-HBV humanized antibody162 (which is described in detail in Chinese Patent Application201610879693.5).

In certain embodiments, the antibody or antigen-binding fragment thereofof the present invention binds to aa121-124 of HBsAg with higheraffinity at neutral pH than at acidic pH.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 andHCDR3, which has one or more of the following characteristics:

(i) HCDR1 has at least one amino acid (for example, 1, 2, 3, 4 or 5amino acids) replaced with histidine as compared with a sequence shownin SEQ ID NO: 11;

(ii) HCDR2 has at least one amino acid (for example, 1, 2, 3, 4 or 5amino acids) replaced with histidine as compared with a sequence shownin SEQ ID NO: 12; and/or,

(iii) HCDR3 has at least one amino acid (for example, 1, 2, 3, 4 or 5amino acids) replaced with histidine as compared with a sequence shownin SEQ ID NO: 13.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises a heavy chain variable region (VL) comprising LCDR1, LCDR2 andLCDR3, which has one or more of the following characteristics:

(i) LCDR1 has at least one amino acid (for example, 1, 2, 3, 4 or 5amino acids) replaced with histidine as compared with a sequence shownin SEQ ID NO: 14;

(ii) LCDR2 has at least one amino acid (for example, 1, 2, 3, 4 or 5amino acids) replaced with histidine as compared with a sequence shownin SEQ ID NO: 15; and/or,

(iii) LCDR3 has at least one amino acid (for example, 1, 2, 3, 4 or 5amino acids) replaced with histidine as compared with a sequence shownin SEQ ID NO: 16.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises:

(a) a heavy chain variable region (VH) comprising the following 3 CDRs:

(i) HCDR1 with a sequence of X₁X₂YHX₃N (SEQ ID NO: 26), wherein X₁ isselected from Y or H, X₂ is selected from G or R, X₃ is selected from Wor Y;

(ii) HCDR2 with a sequence of YIX₄X₅DGSVX₆YNPSLEN (SEQ ID NO: 27),wherein X₄ is selected from S, N or H, X₅ is selected from Y or H, X₆ isselected from L, H or Q; and

(iii) HCDR3 with a sequence of GFDH (SEQ ID NO: 13); and/or,

(b) a light chain variable region (VL) comprising the following 3 CDRs:

(iv) LCDR1 with a sequence of RSSQSLVHSYGDX₇YLH (SEQ ID NO: 28), whereinX₇ is selected from T or N;

(v) LCDR2 with a sequence of KVSNRFS (SEQ ID NO: 15); and

(vi) LCDR3 with a sequence of SQNTHX₈PYT (SEQ ID NO: 29), wherein X₈ isselected from V, L or H.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises:

(a) a heavy chain variable region (VH) comprising the following 3 CDRs:

(i) HCDR1, which is composed of a sequence selected from the following:SEQ ID NOs: 17, 21, 24;

(ii) HCDR2, which is composed of a sequence selected from: SEQ ID NOs:18, 20, 22, 12; and

(iii) HCDR3, which is composed of a sequence shown in SEQ ID NO: 13;and/or,

(b) a light chain variable region (VL) comprising the following 3 CDRs:

(iv) LCDR1, which is composed of a sequence selected from the following:SEQ ID NOs: 14, 25;

(v) LCDR2, which is composed of a sequence shown in SEQ ID NO: 15; and

(vi) LCDR3, which is composed of a sequence selected from the following:SEQ ID NOs: 19, 16, 23.

In certain embodiments, X₁ is selected from H, X₂ is selected from G, X₃is selected from W or Y, X₄ is selected from S or H, X₅ is selected fromY, X₆ is selected from L or H, X₇ is selected from T or N, X₈ isselected from V, L or H.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises:

(a) a heavy chain variable region (VH) comprising the following 3 CDRs:

(i) HCDR1, which is composed of a sequence selected from the following:SEQ ID NOs: 17, 24;

(ii) HCDR2, which is composed of a sequence selected from: SEQ ID NOs:18, 12; and

(iii) HCDR3, which is composed of a sequence shown in SEQ ID NO: 13;and/or,

(b) a light chain variable region (VL) comprising the following 3 CDRs:

(iv) LCDR1, which is composed of a sequence selected from the following:SEQ ID NOs: 14, 25;

(v) LCDR2, which is composed of a sequence shown in SEQ ID NO: 15; and

(vi) LCDR3, which is composed of a sequence selected from the following:SEQ ID NOs: 19, 16, 23.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises:

(1) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 21,HCDR2 shown in SEQ ID NO: 22, HCDR3 shown in SEQ ID NO: 13; and, a VLcomprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 14, LCDR2shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 23;

(2) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 17,HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO: 13; and, a VLcomprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 14, LCDR2shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 19;

(3) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 17,HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 13; and, a VLcomprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 14, LCDR2shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 16;

(4) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 24,HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 13; and, a VLcomprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 25, LCDR2shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 16;

(5) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 17,HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 13; and, a VLcomprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 25, LCDR2shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 23; or

(6) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 17,HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 13; and, a VLcomprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 25, LCDR2shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 16.

In certain embodiments, the antibody or antigen-binding fragment thereoffurther comprises a framework region of a human immunoglobulin (forexample, a framework region contained in an amino acid sequence encodedby a human germline antibody gene), and the framework region optionallycomprises one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10)back mutations from human residues to murine residues.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain framework region contained in an amino acidsequence encoded by a human heavy chain germline gene, and/or a lightchain framework region contained in an amino acid sequence encoded by ahuman light chain germline gene.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain framework region contained in an amino acidsequence encoded by human heavy chain germline gene 4-28-02 (SEQ ID NO:38), and a light chain framework region contained in an amino acidsequence encoded by human light chain germline gene 2D-28-01 (SEQ ID NO:39), and the heavy chain framework region and/or the light chainframework region optionally comprises one or more (for example, 1, 2, 3,4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to murineresidues.

In certain embodiments, the VH of the antibody or antigen-bindingfragment thereof comprises: VH FR1 as shown in SEQ ID NO: 30, VH FR2 asshown in SEQ ID NO: 31, VH FR3 as shown in SEQ ID NO: 32, and VH FR4shown in SEQ ID NO: 33.

In some embodiments, the VL of the antibody or antigen-binding fragmentthereof comprises: VL FR1 as shown in SEQ ID NO: 34, VL FR2 as shown inSEQ ID NO: 35, VL FR3 as shown in SEQ ID NO: 36, and VL FR4 shown in SEQID NO: 37.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises:

(a) a heavy chain variable region (VH), which comprises an amino acidsequence selected from the following:

(i) a sequence shown in any one of SEQ ID NOs: 3, 5, 6, 8;

(ii) a sequence with substitution, deletion or addition of one orseveral amino acids (for example, substitution, deletion or addition of1, 2, 3, 4 or 5 amino acids) as compared with a sequence shown in anyone of SEQ ID NOs: 3, 5, 6, 8; or

(iii) a sequence with a sequence identity of at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% as compared with a sequence shown in any one of SEQ ID NOs: 3, 5,6, 8;

and

(b) a light chain variable region (VL), which comprises an amino acidsequence selected from the following:

(iv) a sequence shown in any one of SEQ ID NOs: 4, 2, 7, 9, 10;

(v) a sequence with substitution, deletion or addition of one or severalamino acids (for example, substitution, deletion or addition of 1, 2, 3,4 or 5 amino acids) as compared with a sequence shown in any one of SEQID NOs: 4, 2, 7, 9, 10; or

(vi) a sequence with a sequence identity of at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% as compared with a sequence shown in any one of SEQ ID NOs: 4, 2,7, 9, 10.

Preferably, the substitution described in (ii) or (v) is a conservativesubstitution.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises:

(1) a VH with a sequence shown in SEQ ID NO: 3 and a VL with a sequenceshown in SEQ ID NO: 4;

(2) a VH with a sequence shown in SEQ ID NO: 5 and a VL with a sequenceshown in SEQ ID NO: 2;

(3) a VH with a sequence shown in SEQ ID NO: 6 and a VL with a sequenceshown in SEQ ID NO: 7;

(4) a VH with a sequence shown in SEQ ID NO: 8 and a VL with a sequenceshown in SEQ ID NO: 9;

(5) a VH with a sequence shown in SEQ ID NO: 3 and a VL with a sequenceshown in SEQ ID NO: 10; or

(6) a VH with a sequence shown in SEQ ID NO: 3 and a VL with a sequenceshown in SEQ ID NO: 9.

In certain embodiments, the antibody or antigen-binding fragment thereoffurther comprises a constant region derived from a human immunoglobulin.

In certain embodiments, the heavy chain of the antibody orantigen-binding fragment thereof comprises a heavy chain constant regionderived from a human immunoglobulin (for example, IgG1, IgG2, IgG3, orIgG4), and the light chain of the antibody or antigen-binding fragmentthereof comprises a light chain constant region derived from a humanimmunoglobulin (for example, κ or λ).

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises:

(a) a heavy chain constant region (CH) of a human immunoglobulin or avariant thereof, wherein the variant has substitution, deletion oraddition of one or more amino acids or any combination thereof (forexample, substitution, deletion or addition of at most 20, at most 15,at most 10, or at most 5 amino acids or any combination thereof; forexample, substitution, deletion or addition of 1, 2, 3, 4 or 5 aminoacids or any combination thereof) as compared with a wild-type sequencefrom which it is derived; and/or

(b) a light chain constant region (CL) of a human immunoglobulin or avariant thereof, wherein the variant has substitution, deletion oraddition of one or more amino acids or any combination thereof (forexample, substitution, deletion or addition of at most 20, at most 15,at most 10, or at most 5 amino acids or any combination thereof; forexample, substitution, deletion or addition of 1, 2, 3, 4 or 5 aminoacids or any combination thereof) as compared with a wild-type sequencefrom which it is derived.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises a human IgG1 or IgG4 heavy chain constant region. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesa heavy chain constant region (CH) as shown in SEQ ID NO: 40.

In certain embodiments, the antibody or antigen-binding fragment thereofof the present invention comprises a variant of a heavy chain constantregion (CH) of a human immunoglobulin, in which the variant has anenhanced affinity to hFcRn or mFcγRII at neutral pH (for example, pH7.4) as compared with a wild-type sequence from which it is derived. Insuch embodiments, the variant generally has substitution of at least oneamino acid as compared with a wild-type sequence from which it isderived.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises a variant of a human IgG1 heavy chain constant region, inwhich the variant has the following substitutions as compared to awild-type sequence from which it is derived: (i) M252Y, N286E, N434Y;or, (ii) K326D, L328Y; wherein the above-mentioned amino acid positionsare positions according to the Kabat numbering system. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesa heavy chain constant region (CH) as shown in SEQ ID NO: 42 or 43.

In certain embodiments, the light chain constant region is a κ lightchain constant region. In certain embodiments, the antibody orantigen-binding fragment thereof comprises a light chain constant region(CL) as shown in SEQ ID NO: 41.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises:

(1) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shownin SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO:4 and a CL shown in SEQ ID NO: 41;

(2) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shownin SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO:4 and a CL shown in SEQ ID NO: 41;

(3) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shownin SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO:4 and a CL shown in SEQ ID NO: 41;

(4) a heavy chain comprising a VH shown in SEQ ID NO: 5 and a CH shownin SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO:2 and a CL shown in SEQ ID NO: 41;

(5) a heavy chain comprising a VH shown in SEQ ID NO: 5 and a CH shownin SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO:2 and a CL shown in SEQ ID NO: 41;

(6) a heavy chain comprising a VH shown in SEQ ID NO: 5 and a CH shownin SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO:2 and a CL shown in SEQ ID NO: 41;

(7) a heavy chain comprising a VH shown in SEQ ID NO: 6 and a CH shownin SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO:7 and a CL shown in SEQ ID NO: 41;

(8) a heavy chain comprising a VH shown in SEQ ID NO: 6 and a CH shownin SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO:7 and a CL shown in SEQ ID NO: 41;

(9) a heavy chain comprising a VH shown in SEQ ID NO: 6 and a CH shownin SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO:7 and a CL shown in SEQ ID NO: 41;

(10) a heavy chain comprising a VH shown in SEQ ID NO: 8 and a CH shownin SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO:9 and a CL shown in SEQ ID NO: 41;

(11) a heavy chain comprising a VH shown in SEQ ID NO: 8 and a CH shownin SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO:9 and a CL shown in SEQ ID NO: 41;

(12) a heavy chain comprising a VH shown in SEQ ID NO: 8 and a CH shownin SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO:9 and a CL shown in SEQ ID NO: 41;

(13) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shownin SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO:10 and a CL shown in SEQ ID NO: 41;

(14) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shownin SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO:10 and a CL shown in SEQ ID NO: 41;

(15) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shownin SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO:10 and a CL shown in SEQ ID NO: 41;

(16) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shownin SEQ ID NO: 40, and a light chain comprising a VL shown in SEQ ID NO:9 and a CL shown in SEQ ID NO: 41;

(17) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shownin SEQ ID NO: 47, and a light chain comprising a VL shown in SEQ ID NO:9 and a CL shown in SEQ ID NO: 41; or

(18) a heavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shownin SEQ ID NO: 48, and a light chain comprising a VL shown in SEQ ID NO:9 and a CL shown in SEQ ID NO: 41.

Preparation of Antibody

The antibody of the present invention can be prepared by various methodsknown in the art, for example, obtained by genetic engineeringrecombination technology. For example, DNA molecules encoding the heavychain and light chain genes of the antibody of the present invention areobtained by chemical synthesis or PCR amplification. The resulting DNAmolecule is inserted into an expression vector and then transfected intoa host cell. Then, the transfected host cell is cultured under specificconditions, and the antibody of the present invention is expressed.

The antigen-binding fragment of the present invention can be obtained byhydrolyzing a complete antibody molecule (see Morimoto et al., J.Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science229:81 (1985)). In addition, these antigen-binding fragments can also bedirectly produced by recombinant host cells (reviewed in Hudson, Curr.Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000))). For example, Fab′ fragments can be obtained directlyfrom host cells; Fab′ fragments can be chemically coupled to formF(ab′)₂ fragments (Carter et al., Bio/Technology, 10: 163-167 (1992)).In addition, Fv, Fab or F(ab′)₂ fragments can also be directly isolatedfrom a recombinant host cell culture medium. Those of ordinary skill inthe art are fully aware of other techniques for preparing theseantigen-binding fragments.

Therefore, in another aspect, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence encoding theantibody or antigen-binding fragment thereof of the present invention,or heavy chain variable region and/or light chain variable regionthereof. In certain preferred embodiments, the isolated nucleic acidmolecule encodes the antibody or antigen-binding fragment thereof of thepresent invention, or heavy chain variable region and/or light chainvariable region thereof.

In another aspect, the present invention provides a vector (for example,a cloning vector or an expression vector) comprising the isolatednucleic acid molecule of the present invention. In certain preferredembodiments, the vector of the present invention is, for example,plasmid, cosmid, bacteriophage and the like.

In another aspect, the present invention provides a host cell comprisingthe isolated nucleic acid molecule of the present invention or thevector of the present invention. Such host cell includes, but is notlimited to, prokaryotic cell such as E. coli cell, and eukaryotic cellsuch as yeast cell, insect cell, plant cell and animal cell (forexample, mammalian cell, such as mouse cell, human cell, etc.). Incertain preferred embodiments, the host cell of the present invention isa mammalian cell, such as CHO (for example, CHO-K1, CHO-S, CHO DG44).

In another aspect, a method for preparing the antibody orantigen-binding fragment thereof of the present invention is provided,which comprises culturing the host cell of the present invention underconditions that allow expression of the antibody or antigen-bindingfragment thereof, and recovering the antibody or antigen-bindingfragment thereof from the cultured host cell culture.

Derived Antibody

The antibody or antigen-binding fragment thereof of the presentinvention can be derivatized, for example linked to another molecule(for example, another polypeptide or protein). Generally, thederivatization (for example, labeling) of the antibody orantigen-binding fragment thereof will not adversely affect its bindingto HBsAg. Therefore, the antibody or antigen-binding fragment thereof ofthe present invention is also intended to include such derivatizedforms. For example, the antibody or antigen-binding fragment of thepresent invention can be functionally linked (by chemical coupling, genefusion, non-covalent linkage or other means) to one or more othermolecular groups, such as another antibody (for example, to form abispecific antibody), detection reagent, pharmaceutical reagent, and/orprotein or polypeptide capable of mediating the antibody orantigen-binding fragment to bind to another molecule (for example,avidin or polyhistidine tag).

Therefore, in certain embodiments, the antibody of the present inventionor antigen-binding fragment thereof is labeled. In some embodiments, theantibody or antigen-binding fragment thereof of the present inventionbears a detectable label, such as enzyme, radionuclide, fluorescent dye,luminescent substance (for example, chemiluminescent substance), orbiotin. The detectable label of the present invention can be anysubstance that can be detected by fluorescence, spectroscopy,photochemistry, biochemistry, immunology, electrical, optical orchemical means. Such labels are well known in the art, examples of whichinclude, but are not limited to, enzyme (for example, horseradishperoxidase, alkaline phosphatase, (β-galactosidase, urease, glucoseoxidase, etc.), radioactive nuclide (for example, ³H, ¹²⁵I, ³⁵S, ¹⁴C or³²P), fluorescent dye (for example, fluorescein isothiocyanate (FITC),fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin(PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (forexample, Cy7, Alexa 750)), luminescent substance (for example,chemiluminescent substance, such as acridine ester compound), magneticbeads (for example, Dynabeads®), calorimetric marker such as colloidalgold or colored glass or plastic (e.g. polystyrene, polypropylene,latex, etc.) beads, and biotin used to ligate avidin (for example,streptavidin) modified by the above-mentioned marker. In certainembodiments, such label can be suitable for immunological detection (forexample, enzyme-linked immunoassay, radioimmunoassay, fluorescentimmunoassay, chemiluminescence immunoassay, etc.). In certainembodiments, the detectable label as described above can be ligated tothe antibody or antigen-binding fragment thereof of the presentinvention through a linker of different length to reduce potentialsteric hindrance.

Pharmaceutical Composition and Therapeutic Use

The antibody or antigen-binding fragment thereof of the presentinvention can be used for the prevention or treatment of an HBVinfection in a subject (for example, a human) or a disease associatedwith HBV infection (for example, hepatitis B), for neutralizing in vitroor in a subject (for example, a human) a virulence of HBV, for reducinga serum level of HBV DNA and/or HBsAg in a subject (for example, ahuman), and for activating a humoral immune response to HBV in a subject(for example, a patient with chronic HBV infection or chronic hepatitisB).

Therefore, in another aspect, the present invention provides apharmaceutical composition, which comprises the antibody orantigen-binding fragment thereof of the present invention, and apharmaceutically acceptable carrier and/or excipient. The pharmaceuticalcomposition of the present invention may also comprises an additionalpharmaceutically active agent. In certain embodiments, the additionalpharmaceutically active agent is a drug used to prevent or treat an HBVinfection or a disease associated with HBV infection (for example,hepatitis B), for example, interferon drug, such as interferon orpegylated interferon.

In another aspect, the present inveiton provides a use of the antibodyor antigen-binding fragment thereof of the present invention or thepharmaceutical composition of the present invention in the manufactureof a medicament for the prevention and/or treatment of an HBV infection(for example, a human) or a disease associated with HBV infection (forexample, hepatitis B) in a subject, for neutralizing a virulence of HBVin vitro or in a subject (for example, a human), for reducing a serumlevel of HBV DNA and/or HBsAg in a subject (for example, a human),and/or for activating a humoral immune response to HBV in a subject (forexample, a patient with chronic HBV infection or chronic hepatitis B).

In another aspect, the present invention provides a method forpreventing or treating an HBV infection or a disease associated with HBVinfection (for example, hepatitis B) in a subject (for example, ahuman), for neutralizing a virulence of HBV in vivo or in a subject (forexample, a human), for reducing a serum level of HBV DNA and/or HBsAg ina subject (for example, a human), and/or for activating a humoral immuneresponse to HBV in a subject (for example, a patient with chronic HBVinfection or chronic hepatitis B), the method comprises administering aneffective amount of the antibody or antigen-binding fragment thereofaccording to the present invention or the pharmaceutical compositionaccording to the present invention to a subject in need thereof.

The drugs and pharmaceutical compositions provided by the presentinvention can be used alone or in combination, and can also be used incombination with other pharmaceutically active agents (for example,other antiviral agents, such as interferon drugs, such as interferon orpegylated interferon).

The antibody or antigen-binding fragment thereof of the presentinvention or the pharmaceutical composition of the present invention canbe administered by a traditional route of administration, including butnot limited to oral, buccal, sublingual, ocular, topical, parenteral,rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical,topical (e.g., powder, ointment or drops), or nasal route. The antibodyor antigen-binding fragment thereof of the present invention can beadministered by various methods known in the art. However, for manytherapeutic applications, the preferred route/mode of administration isparenteral administration (for example, intravenous injection,subcutaneous injection, intraperitoneal injection, intramuscularinjection). The skilled person should understand that the route and/ormode of administration will vary according to the intended purpose. In apreferred embodiment, the antibody or antigen-binding fragment thereofof the present invention is administered by intravenous infusion orinjection.

The antibody or antigen-binding fragment thereof of the presentinvention or the pharmaceutical composition of the present invention canbe formulated into a variety of dosage forms, such as liquid, semisolid,and solid forms, for example, solution (e.g. injection), dispersion orsuspension, tablet, powder, granule, emulsion, pill, syrup, powder,liposome, capsule and suppository. The preferred dosage form depends onthe intended mode of administration and therapeutic use.

For example, one preferred dosage form is an injection. Such aninjection may be a sterile injectable solution. For example, a sterileinjectable solution can be prepared by the following method: a necessarydose of the antibody or an antigen binding fragment thereof according tothe invention is incorporated into a suitable solvent, and optionally,other expected ingredients (including, but not limited to, a pHregulator, a surfactant, an adjuvant, an ionic strength enhancer, anisotonic agent, a preservative, a diluent, or any combination thereof)are incorporated simultaneously, and then filtered sterilization iscarried out. In addition, the sterile injectable solution can beprepared into a sterile powder (for example, by vacuum drying or freezedrying) for the convenience of storage and use. Such sterile powder canbe dispersed in a suitable vehicle before use, such as sterilepyrogen-free water.

Another preferred dosage form is a dispersion. A dispersion can beprepared by the following method: the antibody or an antigen bindingfragment thereof according to the invention is incorporated in a sterilevehicle comprising a basic dispersion medium and optionally, otherexpected ingredients (including, but not limited to, a pH regulator, asurfactant, an adjuvant, an ionic strength enhancer, an isotonic agent,a preservative, a diluent, or any combination thereof). In addition, anabsorption delaying agent can also be incorporated in a dispersion, suchas monostearate salt and gelatin, in order to obtain an expectedpharmacokinetic property.

Another preferred dosage form is an oral solid dosage form, includingcapsule, tablet, powder, granule, and the like. Such a solid dosage formgenerally comprises at least one of: (a) inert drug excipient (orvehicle), such as sodium citrate and calcium phosphate; (b) filler, suchas starch, lactose, sucrose, mannose and silicic acid; (c) binder, suchas carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone,sucrose and arabic gum; (d) wetting agent, such as glycerol; (e)disintegrating agent, such as agar, calcium carbonate, potato or tapiocastarch; (f) retarder, such as olefin; (g) absorption enhancer, such asquaternary ammonium compound; (h) humectant, such as cetyl alcohol andglyceryl monostearate; (i) adsorbent, such as kaolin and bentonite; (j)lubricant, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycol, sodium dodecyl sulfate, or any combination thereof.In the case of tablet and capsule dosage forms, a buffer can also becomprised.

In addition, a release rate modifier (i.e. an agent capable of changingdrug release rate) may also be added to an oral solid dosage form, inorder to obtain a modified release or pulsed release dosage form. Such arelease rate modifier includes, but is not limited to carboxypropylmethylcellulose, methylcellulose, carboxymethyl cellulose sodium, ethylcellulose, cellulose acetate, polyethylene oxide, xanthan gum,isoacrylic amino copolymer, hydrogenated flavoring oil, carnauba wax,paraffin, cellulose acetate phthalate, carboxypropyl methylcellulosephthalate, methacrylic acid copolymer, or any combination thereof. Amodified release or pulsed release dosage form may comprise one or agroup of release rate modifiers.

Another preferred dosage form is an oral liquid dosage form, includingemulsion, solution, suspension, syrup, and the like. In addition toactive ingredients, such an oral liquid dosage form may further compriseinert solvents commonly used in the art, for example water or othersolvents, such as ethyl alcohol, isopropanol, propylene glycol,1,3-butylene glycol, oil (such as cotton seed oil, peanut oil, corn oil,olive oil, flavoring oil and sesame oil), glycerol, polyethylene glycoland sorbitan fatty acid ester, and any combination thereof. In additionto these inert solvents, such an oral liquid dosage form may furthercomprise humectant, emulsifying agent, suspending agent, sweeteningagent, flavoring agent, fragrant agent, and the like.

In addition, the antibody or an antigen binding fragment thereofaccording to the invention may be present in a unit dosage form in apharmaceutical composition, for the convenience of administration. Thepharmaceutical composition according to the invention should be sterile,and stable under the conditions of manufacture and storage conditions.

The medicament and pharmaceutical composition provided in the inventionmay be used alone or in combination, or may be used in combination withan additional pharmaceutically active agent (for example, otherantiviral agents, e.g. interferon-type agents, such as interferon orpegylated interferon). In some preferred embodiments, the antibody or anantigen binding fragment thereof according to the invention is used incombination with other antiviral agent(s), in order to prevent and/ortreat a disease associated with HBV infection. The antibody or anantigen binding fragment thereof according to the invention and suchantiviral agent(s) can be administered simultaneously, separately orsequentially. Such antiviral agent(s) include, but are not limited to,interferon-type agents, ribavirin, adamantane, hydroxyurea, IL-2, L-12and pentacarboxy cytosolic acid, etc.

The pharmaceutical composition according to the invention may comprise“a therapeutically effective amount” or “a prophylactically effectiveamount” of the antibody or an antigen binding fragment thereof accordingto the invention. “A prophylactically effective amount” refers to anamount that is sufficient to prevent, suppress or delay the developmentof a disease (such as HBV infection or a disease associated with HBVinfection). “A therapeutically effective amount” refers to an amountthat is sufficient to cure or at least partially suppress a disease andits complications in a patient with the disease. The therapeuticallyeffective amount of the antibody or an antigen binding fragment thereofaccording to the invention may vary depending on the following factors:the severity of a disease to be treated, general state of the immunesystem in a patient, general conditions of a patient such as age, weightand gender, administration modes of drugs, additional therapies usedsimultaneously, and the like.

A dosage regimen can be adjusted to provide an optimal desired effect(for example, a therapeutic or prophylactic effect). For example, asingle dose may be administered, or multiple doses may be administeredwithin a period of time, or the dose can be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.

For the antibody or antigen binding fragment thereof according to theinvention, an exemplary and non-limiting range for a therapeutically orprophylactically effective amount is from 0.025 to 50 mg/kg, morepreferably from 0.1 to 50 mg/kg, more preferably 0.1-25 mg/kg, 0.1-10mg/kg. It should be noticed that a dose can vary depending on the typeand severity of a disease to be treated. In addition, a person skilledin the art understands that for any specific patient, specific dosageregimen should be adjusted over time depending on the patient's need andthe professional evaluation made by a doctor; the dose range providedhere is only provided for the purpose of exemplification, rather thandefining the use or scope of the pharmaceutical composition according tothe invention.

Kit and Detection Use

The antibody or antigen-binding fragment thereof of the presentinvention can specifically bind to HBsAg, so that it can be used todetect the presence or level of HBsAg in a sample.

Therefore, in another aspect, the present invention provides a kitcomprising the antibody or antigen-binding fragment thereof of thepresent invention. In some embodiments, the antibody or antigen-bindingfragment thereof of the present invention bears a detectable label. Inother embodiments, the kit further comprises a second antibody, whichspecifically recognizes the antibody or antigen-binding fragment thereofof the present invention. Preferably, the second antibody furthercomprises a detectable label. Such detectable labels are well known tothose skilled in the art, and include, but are not limited to,radioisotope, fluorescent substance, luminescent substance, coloredsubstance and enzyme (for example, horseradish peroxidase) and the like.

In another aspect, the present invention provides a method for detectingthe presence or level of HBsAg protein in a sample, which comprises:using the antibody or antigen-binding fragment thereof of the presentinvention. In some embodiments, the antibody or antigen-binding fragmentthereof of the present invention further comprises a detectable label.In other embodiments, the method further comprises using a secondantibody carrying a detectable label to detect the antibody orantigen-binding fragment thereof of the present invention. The methodcan be used for diagnostic purposes, or for non-diagnostic purposes (forexample, the sample is a cell sample, not a sample from a patient).

In some embodiments, the method comprises: (1) contacting the samplewith the antibody or antigen-binding fragment thereof of the presentinvention; (2) detecting the formation of a complex between the antibodyor antigen-binding fragment thereof and HBsAg protein or detecting anamount of the complex. The formation of the complex indicates thepresence of HBsAg protein and/or HBV.

In another aspect, the present invention provides a method fordiagnosing whether a subject is infected with HBV, which comprises:using the antibody or antigen-binding fragment thereof of the presentinvention to detect the presence of HBsAg protein in a sample from thesubject. In some embodiments, the antibody or antigen-binding fragmentthereof of the present invention further comprises a detectable label.In other embodiments, the method further comprises using a secondantibody carrying a detectable label to detect the antibody orantigen-binding fragment thereof of the present invention.

In another aspect, there is provided a use of the antibody orantigen-binding fragment thereof of the present invention in themanufacture of a kit for detecting the presence or level of HBsAgprotein in a sample, or for diagnosing whether a subject is infectedwith HBV.

Definition of Terms

In the present invention, unless otherwise specified, the scientific andtechnical terms used herein have the meanings commonly understood bythose skilled in the art. Moreover, the cell culture, biochemistry,nucleic acid chemistry, immunology laboratory and other operating stepsused in this article are all routine steps widely used in thecorresponding fields. At the same time, in order to better understandthe present invention, definitions and explanations of related terms areprovided below.

As used herein, the term “antibody” refers to an immunoglobulin moleculetypically composed of two pairs of polypeptide chains, each pair havinga light chain (LC) and a heavy chain (HC). Antibody light chains can beclassified into κ (kappa) and λ (lambda) light chains. Heavy chains canbe classified as μ, δ, γ, α, or ε, and the isotypes of antibody aredefined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the lightand heavy chains, the variable and constant regions are connected by a“J” region of about 12 or more amino acids, and the heavy chain alsocomprises a “D” region of about 3 or more amino acids. Each heavy chainis composed of a heavy chain variable region (VH) and a heavy chainconstant region (CH). The heavy chain constant region is composed of 3domains (CH1, CH2, and CH3). Each light chain is composed of a lightchain variable region (VL) and a light chain constant region (CL). Thelight chain constant region is composed of a domain CL. The constantdomain does not directly participate in the binding of antibody andantigen, but exhibits a variety of effector functions, such as mediatingthe binding of immunoglobulin to a host tissue or factor, includingvarious cells of immune system (for example, effector cells) and thefirst component of classical complement system (C1q). The VH and VLregions can also be subdivided into hypervariable regions (calledcomplementarity determining regions (CDRs)), interspersed withrelatively conservative regions called framework regions (FRs). Each VHand VL is composed of 3 CDRs and 4 FRs arranged in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to thecarboxy terminus. The variable regions (VH and VL) of each heavychain/light chain pair form antigen binding site respectively. Theassignment of amino acids in each region or domain can follow thedefinitions of Kabat, Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989)Nature 342:878-883.

As used herein, the term “complementarity determining region” or “CDR”refers to amino acid residues in a variable region of an antibody thatare responsible for antigen binding. Each of the variable regions of theheavy chain and the light chain contains three CDRs, named CDR1, CDR2,and CDR3. The precise boundaries of these CDRs can be defined accordingto various numbering systems known in the art, for example, according tothe Kabat numbering system (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991), Chothia numbering system(Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989)Nature 342:878-883) or IMGT numbering system (Lefranc et al. al., Dev.Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilledin the art will easily identify the CDRs defined by each numberingsystem. Moreover, the correspondence between different numbering systemsis well known to those skilled in the art (for example, see Lefranc etal., Dev. Comparat. Immunol. 27:55-77, 2003).

In the present invention, the CDRs contained in the antibody orantigen-binding fragment thereof of the present invention can bedetermined according to various numbering systems known in the art. Incertain embodiments, the CDRs contained in the antibody orantigen-binding fragment thereof of the present invention are preferablydetermined by the Kabat, Chothia or IMGT numbering system. In certainembodiments, the CDRs contained in the antibody or antigen-bindingfragment thereof of the present invention are preferably determined bythe Kabat numbering system.

As used herein, the term “framework region” or “FR” residues refers tothose amino acid residues in a variable region of an antibody other thanthe CDR residues as defined above.

The term “antibody” is not limited by any specific method for producingthe antibody. For example, it comprises recombinant antibody, monoclonalantibody, and polyclonal antibody. The antibody may be an antibody ofdifferent isotype, for example, IgG (for example, IgG1, IgG2, IgG3 orIgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

As used herein, the term “antigen-binding fragment” of antibody refersto a polypeptide comprising a fragment of a full-length antibody thatretains the ability to specifically bind to the same antigen to whichthe full-length antibody binds, and/or competes with the full-lengthantibody to specifically bind to the antigen, which is also called“antigen binding portion”. See generally, Fundamental Immunology, Ch. 7(Paul, W., ed., 2nd edition, Raven Press, NY (1989), which isincorporated herein by reference in its entirety for all purposes.Antigen-binding fragment of antibody can be produced by recombinant DNAtechnology or by the enzymatic or chemical cleavage of the intactantibody. Non-limiting examples of antigen-binding fragment include Fab,Fab′, F(ab′)₂, Fd, Fv, complementarity determining region (CDR)fragments, scFv, diabody, single domain antibody, chimeric antibody,linear antibody, nanobody (technology from Domantis), probody and suchpolypeptides which comprise at least a portion of the antibody that isenough to confer a specific antigen-binding capacity to thepolypeptides. Engineered antibody variants are reviewed in Holliger etal., 2005; Nat Biotechnol, 23: 1126-1136.

As used herein, the term “full-length antibody” refers to an antibodycomposed of two “full-length heavy chains” and two “full-length lightchains.” “full-length heavy chain” refers to a polypeptide composed of aheavy chain variable region (VH), a heavy chain constant region CH1domain, a hinge region (HR), a heavy chain constant region CH2 domainand a heavy chain constant region CH3 domain in the N-terminal toC-terminal direction; and, when the full-length antibody is of the IgEisotype, it optionally also comprises a heavy chain constant region CH4domain. Preferably, the “full-length heavy chain” is a polypeptide chaincomposed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminaldirection. The “full-length light chain” is a polypeptide chain composedof a light chain variable region (VL) and a light chain constant region(CL) in the N-terminal to C-terminal direction. The two pairs offull-length antibody chains are connected by a disulfide bond between CLand CH1 and a disulfide bond between HRs of the two full-length heavychains. The full-length antibody of the present invention can be derivedfrom a single species, such as human; it can also be a chimeric antibodyor a humanized antibody. The full-length antibody of the presentinvention comprises two antigen binding sites formed by VH and VL pairsrespectively, and the two antigen binding sites specificallyrecognize/bind the same antigen.

As used herein, the term “Fd” refers to an antibody fragment composed ofVH and CH1 domains; the term “dAb fragment” refers to an antibodyfragment composed of VH domain (Ward et al., Nature 341:544 546 (1989));the term “Fab fragment” refers to an antibody fragment composed of VL,VH, CL and CH1 domains; the term “F(ab′)₂ fragment” refers to anantibody fragment composed of two Fab fragments connected by a disulfidebridge on the hinge region; the term “Fab′ fragment” refers to afragment obtained by reducing the disulfide bond connecting the twoheavy chain fragments in the F(ab′)₂ fragment, and is composed of anintact light chain and a Fd fragment (consisting of VH and CH1 domains)of heavy chain.

As used herein, the term “Fv” refers to an antibody fragment composed ofa single-arm VL and VH domains of an antibody. Fv fragment is generallyconsidered to be the smallest antibody fragment that can form a completeantigen-binding site. It is generally believed that six CDRs conferantigen-binding specificity to an antibody. However, even one variableregion (e.g., Fd fragment, which contains only three antigen-specificCDRs) can recognize and bind to antigen, although its affinity may belower than the complete binding site.

As used herein, the term “Fc” refers to an antibody fragment that isformed by linking the second, third constant region of a first heavychain of an antibody and the second, third constant region of a secondheavy chain via disulfide bonding. The Fc fragment of an antibody hasmany different functions, but does not participate in antigen binding.

As used herein, the term “scFv” refers to a single polypeptide chaincomprising VL and VH domains, wherein the VL and VH are connected by alinker (see, for example, Bird et al., Science 242:423 -426 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); andPluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburgand Moore eds, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFvmolecules may have the general structure: NH₂-VL-linker-VH-COOH orNH₂-VH-linker-VL-COOH. Suitable prior art linkers consist of repeatedGGGGS amino acid sequences or variants thereof. For example, a linkerhaving the amino acid sequence (GGGGS)₄ can be used, but variantsthereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci.USA 90: 6444-6448). Other linkers that can be used in the presentinvention are described by Alfthan et al. (1995), Protein Eng.8:725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al.(1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol.Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In somecases, there may also be disulfide bonds between the VH and VL of thescFv.

As used herein, the term “diabody” refers to that its VH and VL domainsare expressed on a single polypeptide chain, but the used linker is tooshort to allow pairing between the two domains of the same chain,thereby forcing one domain to pair with the complementary domain ofanother chain and generating two antigen-binding sites (see, forexample, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

Each of the aforementioned antibody fragments maintains the ability tospecifically bind to the same antigen to which the full-length antibodybinds, and/or competes with the full-length antibody to specificallybind to the antigen.

Conventional techniques known to those skilled in the art (for example,recombinant DNA technology or enzymatic or chemical fragmentation) canbe used to obtain from a given antibody (for example, the antibodyprovided by the present invention) the antigen-binding fragments of theantibody (for example, the above-mentioned antibody fragments), and canbe screened for specificity in the same manner by which intactantibodies are screened.

Herein, unless the context clearly dictates otherwise, when the term“antibody” is referred to, it includes not only intact antibody but alsoantigen-binding fragments of the antibody.

As used herein, the term “monoclonal antibody”, “McAb” and “mAb” havethe same meaning and can be used interchangeably. It refers to anantibody or a fragment of an antibody from a population of highlyhomologous antibody molecules, i.e. a population of completely identicalantibody molecules except for natural mutation that may occurspontaneously. A monoclonal antibody has a high specificity for a singleepitope of an antigen. Polyclonal antibody, relative to monoclonalantibody, generally comprises at least two or more different antibodieswhich generally recognize different epitopes on an antigen. In addition,the modifier “monoclonal” merely indicates the character of the antibodyas being obtained from a highly homogeneous population of antibodies,and is not to be construed as requiring production of the antibody byany particular method.

As used herein, the term “chimeric antibody” refers to an antibody thata part of its light chain or/and heavy chain is derived from an antibody(which may be derived from a specific species or belong to a specificantibody class or subclass), and another part of its light chain or/andheavy chain is derived from another antibody (which may be derived fromthe same or different species or belong to the same or differentantibody class or subclass), but in any case, it still retains thebinding activity to the target antigen (U.S. Pat. No. 4,816,567 toCabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)). For example, the term “chimeric antibody” may include suchan antibody (e.g., human-mouse chimeric antibody), in which the heavyand light chain variable regions of the antibody are derived from afirst antibody (e.g., mouse antibody), while the heavy chain and lightchain constant regions of the antibody are derived from a secondantibody (e.g., human antibody). In order to prepare a chimericantibody, the methods known in the art can be used to linkimmunoglobulin variable regions of an immunized animal to humanimmunoglobulin constant regions (see, for example, U.S. Pat. No.4,816,567 to Cabilly et al.). For example, a DNA encoding VH is operablylinked to another DNA molecule encoding the heavy chain constant regionto obtain a full-length heavy chain gene. The sequence of the humanheavy chain constant region gene is known in the art (see, for example,Kabat, E A et al. (1991), Sequences of Proteins of ImmunologicalInterest, Fifth Edition, US Department of Health and Human Services, NIHPublication No. 91-3242), the DNA fragments comprising these regions canbe obtained by standard PCR amplification. The heavy chain constantregion may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constantregion, but is generally preferably an IgG1 or IgG4 constant region. Forexample, the DNA encoding VL is operably linked to another DNA moleculeencoding the light chain constant region CL to obtain a full-lengthlight chain gene (and a Fab light chain gene). The sequence of the humanlight chain constant region gene is known in the art (see, for example,Kabat, EA et al. (1991), Sequences of Proteins of ImmunologicalInterest, Fifth Edition, US Department of Health and Human Services, NTHPublication No. 91-3242), and DNA fragments comprising these regions canbe obtained by standard PCR amplification. The light chain constantregion can be a ϵ or λ, constant region, but is generally preferably a κconstant region.

As used herein, the term “humanized antibody” refers to a geneticallyengineered non-human antibody, whose amino acid sequence has beenmodified to increase homology with the sequence of a human antibody.Generally speaking, all or part of the CDR regions of a humanizedantibody are derived from a non-human antibody (donor antibody), and allor part of the non-CDR regions (for example, variable region FR and/orconstant region) are derived from a human immunoglobulin (receptorantibody). In some embodiments, the CDR regions of the humanizedantibody are derived from a non-human antibody (donor antibodies), andall or part of the non-CDR regions (for example, variable region FRand/or constant regions) are derived from a human immunoglobulin(receptor antibody). The humanized antibody generally retains theexpected properties of the donor antibody, including, but not limitedto, antigen specificity, affinity, reactivity, etc. The donor antibodymay be a mouse, rat, rabbit, or non-human primate (for example,cynomolgus monkey) antibody with desired properties (for example,antigen specificity, affinity, reactivity, etc.). In order to preparethe humanized antibody, the methods known in the art can be used toinsert the CDR regions of the immunized animal into the human frameworksequences (see U.S. Pat. No. 5,225,539 to Winter; U.S. Pat. No.5,530,101 to Queen et al.; U.S. PAt. Nos. 5,585,089; 5,693,762 and6,180,370; and Lo, Benny, K C, editor, in Antibody Engineering: Methodsand Protocols, volume 248, Humana Press, New Jersey, 2004).

As used herein, the term “germline antibody gene” or “germline antibodygene segment” refers to a sequence present in the genome of an organismencoding immunoglobulin, which has not undergone a maturation processthat can lead to genetic rearrangements and mutations for expression ofa particular immunoglobulin. In the present invention, the expression“heavy chain germline gene” refers to an germline antibody gene or genefragment encoding an immunoglobulin heavy chain, which includes V gene(variable), D gene (diversity), J gene (joining) and C gene (constant);similarly, the expression “light chain germline gene” refers to angermline antibody gene or gene fragment encoding an immunoglobulin lightchain, which includes V gene (variable), J gene (joining), and C gene(constant). In the present invention, the amino acid sequence encoded bythe germline antibody gene or the germline antibody gene fragment isalso referred to as “germline sequence”. The germline antibody gene orgermline antibody gene fragment and their corresponding germlinesequences are well known to those skilled in the art and can be obtainedor queried from professional databases (e.g., IMGT, unswag, NCBI orVBASE2).

As used herein, the term “specific binding” refers to a non-randombinding reaction between two molecules, such as the reaction between anantibody and an antigen to which it is directed. The strength oraffinity of a specific binding interaction can be expressed by anequilibrium dissociation constant (K_(D)) of the interaction. In thepresent invention, the term “K_(D)” refers to a dissociation equilibriumconstant of a specific antibody-antigen interaction, which is used todescribe the binding affinity between the antibody and the antigen. Thesmaller the equilibrium dissociation constant, the tighter theantibody-antigen binding, and the higher the affinity between theantibody and the antigen. The specific binding properties between twomolecules can be measured using methods known in the art, for example,using surface plasmon resonance (SPR) of BIACORE instrument.

As used herein, the expression “binding at a neutral pH with an affinityhigher than that at an acidic pH” or the equivalent expression“pH-dependent binding” refers to that the antibody of the presentinvention has a K_(D) value or EC50 value for binding HBsAg at an acidicpH that is higher than its K_(D) value or EC50 value for binding HBsAgat a neutral pH. The K_(D) can be measured by a technique known in theart, for example, by SPR technique (for example, Biacore). In thepresent invention, the term “EC50” refers to an antibody-antigen halfmaximum effect concentration, that is, an antibody concentrationrequired to reach 50% of the maximum binding effect between a specificantibody-antigen, and it is used to describe the binding capacitybetween the antibody and the antigen. The smaller the EC50, the higherthe binding capacity between the antibody and the antigen. Theantibody-antigen half maximum effect concentration (EC50) can bedetermined using methods known in the art, for example, using anenzyme-linked immunosorbent assay (ELISA) in which an antigen is boundto a solid phase carrier, and the antibody specifically binds to theantigen.

As used herein, “neutralizing antibody” refers to an antibody orantigen-binding fragment thereof that can significantly reduce orcompletely inhibit the virulence (for example, the ability to infectcells) of the target virus. Generally speaking, neutralizing antibodiescan recognize and bind the target virus, and prevent the target virusfrom entering/infecting the subject's cells. The antibody of the presentinvention is a neutralizing antibody.

However, it should be understood that in the present application, thevirus-neutralizing ability of an antibody is not directly equivalent tothe virus-clearing ability of an antibody. As used herein, “neutralizingvirus” means that the virulence of a target virus is neutralized (i.e.the virulence of a target virus is significantly reduced or completelyinhibited) by inhibiting the target virus from entering/infecting thecell of a subject. As used herein, “clearing virus” means that a targetvirus (no matter it infects a cell or not) is eliminated from anorganism, and therefore the organism turns toward the state beforeinfection by the virus (e.g. the serological test result of virus turnsnegative). Therefore, in general, neutralizing antibodies do notnecessarily have virus-clearing ability. However, in the presentapplication, the inventor surprisingly found that the antibodiesaccording to the invention can not only neutralize HBV, but also clearvirus (i.e. can clear HBV DNA and/or HBsAg in vivo, clear HBV andHBV-infected cells in vivo), and therefore have important clinicalvalue.

As used herein, the term “isolated” refers to a state obtained fromnatural state by artificial means. If a certain “isolated” substance orcomponent is present in nature, it is possible because its naturalenvironment changes, or the substance is isolated from naturalenvironment, or both. For example, a certain un-isolated polynucleotideor polypeptide naturally exists in a certain living animal body, and thesame polynucleotide or polypeptide with a high purity isolated from sucha natural state is called isolated polynucleotide or polypeptide. Theterm “isolated” excludes neither the mixed artificial or synthesizedsubstance nor other impure substances that do not affect the activity ofthe isolated substance.

As used herein, the term “vector” refers to a nucleic acid vehicle intowhich a polynucleotide can be inserted. When a vector enables theexpression of a protein encoded by an inserted polynucleotide, thevector is referred to as an expression vector. A vector can beintroduced into a host cell by transformation, transduction ortransfection, so that the genetic material elements carried by thevector can be expressed in the host cell. Vectors are well known tothose skilled in the art and include, but are not limited to: plasmids;phagemids; cosmids; artificial chromosomes, such as yeast artificialchromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derivedartificial chromosomes (PAC); bacteriophages such as λ phage or M13phage and animal viruses. Animal viruses that can be used as vectorsinclude, but are not limited to, retroviruses (including lentiviruses),adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpessimplex virus), poxviruses, baculoviruses, papillomaviruses, papovavirus(e.g., SV40). A vector may comprise a variety of elements that controlexpression, including, but not limited to, promoter sequence,transcription initiation sequence, enhancer sequence, selection element,and reporter gene. In addition, the vector may comprise a replicationinitiation site.

As used herein, the term “host cell” refers to a cell into which avector can be introduced, which includes, but is not limited to,prokaryotic cell such as Escherichia coli or Bacillus subtilis, fungalcell such as yeast cell or Aspergillus, insect cell such as S2Drosophila cell or Sf9, or animal cell such as fibroblast, CHO cell, COScell, NSO cell, HeLa cell, BHK cell, HEK 293 cell or human cell.

As used herein, the term “identity” refers to the match degree betweentwo polypeptides or between two nucleic acids. When two sequences forcomparison have the same monomer sub-unit of base or amino acid at acertain site (e.g., each of two DNA molecules has an adenine at acertain site, or each of two polypeptides has a lysine at a certainsite), the two molecules are identical at the site. The percent identitybetween two sequences is a function of the number of identical sitesshared by the two sequences over the total number of sites forcomparison×100. For example, if 6 of 10 sites of two sequences arematched, these two sequences have an identity of 60%. For example, DNAsequences: CTGACT and CAGGTT share an identity of 50% (3 of 6 sites arematched). Generally, the comparison of two sequences is conducted in amanner to produce maximum identity. Such alignment can be conducted byusing a computer program such as Align program (DNAstar, Inc.) which isbased on the method of Needleman, et al. (J. Mol. Biol. 48:443-453,1970). The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percentage ofidentity between two amino acid sequences can be determined by thealgorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970))which has been incorporated into the GAP program in the GCG softwarepackage (available at http://www.gcg.com), using either a Blossum 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6.

The twenty conventional amino acids involved herein are expressed inroutine manners. See, for example, Immunology-A Synthesis (2nd Edition,E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass.(1991)), which is incorporated herein by reference. In the presentdisclosure, the terms “polypeptide” and “protein” have the same meaningand are used interchangeably. Also in the present disclosure, aminoacids are generally represented by single letter and three letterabbreviations as known in the art. For example, alanine can berepresented by A or Ala. In addition, as used herein, the terms“monoclonal antibody” and “McAb” have the same meaning and can be usedinterchangeably; the terms “polyclonal antibody” and “PcAb” have thesame meaning and can be used interchangeably.

As used herein, the term “a pharmaceutically acceptable carrier and/orexcipient” refers to a carrier and/or excipient pharmacologically and/orphysiologically compatible with a subject and an active agent, which iswell known in the art (see, e.g., Remington's Pharmaceutical Sciences.Edited by Gennaro A R, 19th ed. Pennsylvania: Mack Publishing Company,1995), and includes, but is not limited to a pH adjuster, a surfactant,an adjuvant, an ionic strength enhancer, a diluent, an osmoticpressure-controlling agent, an absorption delaying agent, and apreservative. For example, the pH adjuster includes, but is not limitedto, phosphate buffer. The surfactant includes, but is not limited to,cationic, anionic, or non-ionic surfactant, e.g. Tween-80. The ionicstrength enhancer includes, but is not limited to, sodium chloride. Thepreservative includes, but is not limited to a variety of antibacterialagents and antifungal agents, such as paraben, chlorobutanol, phenol,and sorbic acid. The osmotic pressure-controlling agent includes, but isnot limited to sugar, NaCl and analogs thereof. The absorption delayingagent includes, but is not limited to monostearate and gelatin.

As used herein, the term “prevention/preventing” refers to a method thatis carried out in order to suppress or delay the occurrence of adisease, a disorder or a symptom (such as HBV infection or a diseaseassociated with HBV infection) in a subject. As used herein, the term“treatment/treating” refers to a method that is carried out in order toobtain a beneficial or desired clinical outcome. For the purpose of theinvention, the beneficial or desired clinical outcome includes, but isnot limited to, easing symptom, narrowing the scope of disease,stabilizing (i.e. not aggravating) the state of disease, delaying orslowing the progress of disease, and alleviating symptoms (eitherpartially or completely), no matter detectable or not detectable. Inaddition, “treatment” also refers to a prolonged survival periodcompared to the expected survival period (if no treatment is accepted).In the present application, the antibody according to the invention hasthe ability of neutralizing HBV, and therefore can be used toprevent/protect an unaffected subject or a cell thereof from infectionby HBV. In addition, the antibody according to the invention has theability of clearing HBV (i.e. able to clear HBV DNA and/or HBsAg invivo, clear HBV and cells infected by HBV in vivo), and therefore can beused to treat HBV infection or a disease associated with HBV infectionin an infected subject.

As used herein, the term “subject” refers to a mammal, such as a primatemammal, such as a human.

As used herein, the term “an effective amount” refers to an amount thatis sufficient to achieve or at least partially achieve the expectedeffect. For example, an amount effective for preventing a disease (suchas HBV infection or diseases associated with HBV infection) refers to anamount effective for preventing, suppressing, or delaying the occurrenceof a disease (such as HBV infection or diseases associated with HBVinfection). An effective amount for treating a disease refers to anamount effective for curing or at least partially blocking a disease andits complication in a patient having the disease. The determination ofsuch an effective amount is within the ability of a person skilled inthe art. For example, an amount effective for a therapeutic use dependson severity of a disease to be treated, general state of the immunesystem in a patient, general conditions of a patient, such as age,weight and gender, administration means of drugs, additional therapiesused simultaneously, and the like.

Beneficial Effects of the Present Invention

The antibody of the present invention not only can specificallyrecognize/bind HBsAg, can neutralize the virulence of HBV, can reducethe serum level of HBV DNA and/or HBsAg in the subject, and caneffectively eliminate HBV and HBV-infected cells in the body, but alsohas a significantly enhanced antigen clearance effect and antigensuppression time. It is particularly surprising that it is known in theart that patients with chronic hepatitis B tend to produce immunedepletion (tolerance) against HBV due to high levels of HBsAg in thebody, thereby prolonging the infection, but the antibody of the presentinvention can activate the subject (for example patients with chronicHBV infection, or patients with chronic hepatitis B) to regenerate ahumoral immune response against HBV, thereby increasing the clinicalcure rate. Therefore, the antibody of the present invention isparticularly suitable for preventing and treating HBV infection anddiseases associated with HBV infection (for example, hepatitis B). Inaddition, the antibody of the present invention has pH-dependent antigenbinding properties, and a single molecule of antibody can bind tomultiple molecules of antigens, so that it can also reduce the frequencyand dosage of administration, and has great clinical value.

The embodiments of the present invention will be described in detailbelow in conjunction with the accompanying drawings and examples.However, those skilled in the art will understand that the followingdrawings and examples are only used to illustrate the present invention,but not to limit the scope of the present invention. According to theaccompanying drawings and the following detailed description of thepreferred embodiments, various objects and advantageous aspects of thepresent invention will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the working principle of an antibodywith pH-dependent antigen-binding activity. Human plasma is neutral,with a pH of about 7.4, while the intracellular environment is acidic,with a pH of about 6.0. An antibody with pH-dependent antigen-bindingactivity can bind to an antigen in the plasma, the antigen-antibodycomplex is then internalized into the cell. The pH-dependent antibodywill dissociate from the antigen in the acidic environment of theendosome. The antibody dissociated from the antigen will be captured byFcRn and circulated to the outside of the cell. In the extracelluarneutral environment, the FcRn releases the antibody, and the antibodyreturned to the plasma can bind to other antigen again, therebyrealizing the cycle use of the antibody.

FIG. 2 shows the results of docking of Fab crystal structure based onthe structural analysis of 162 to a short antigen mimic peptide, inwhich the blue structure is the short antigen peptide, and the redstructure is part of the binding region of 162 antibody.

FIG. 3 shows a schematic diagram of the recombinant vector (pCGMT-scFv)encoding the scFv antibody, in which the scFv antibody has a structureof: NH₂-VH-linker-VL-COOH.

FIGS. 4A to 4D show the ELISA results of the phage library displayingthe pH-dependent scFv antibody derived from 162 and the antigen HBsAg.FIG. 4A: the detection results of binding to HBsAg at pH 7.4 and pH 6.0for the phage library derived from 162 after the third round ofscreening, the abscissa represents the phage antibody number, and theordinate represents the OD value. The results show that these singleclones all have strong antigen binding activity and have a significantdecrease in binding activity at pH 6.0. FIG. 4B: the detection resultsof pH-dependent binding to HBsAg for the 13 single clones with highOD_((450/630)) value at pH 7.4 in the third round and showing thelargest difference between OD_((450/630)) values at pH7.4 and pH 6.0,with 8 gradients and 3-fold dilution, in which the abscissa representsthe dilution factor, and the ordinate represents the OD value. Theresults show that the pH-dependent antigen binding effect is betterpresented after the gradient dilution, in which the C32, C27, C26 andC19 show the better performance and C27 molecule has the best effect(the remaining 9 molecules are not shown). FIG. 4C: the detectionresults of binding to HBsAg at pH 7.4 and pH 6.0 for the phage libraryderived from 162 after the fourth round of screening, the abscissarepresents the phage antibody number, and the ordinate represents the ODvalue. FIG. 4D: the detection results of pH-dependent binding to HB sAgfor the 8 single clones with high OD_((450/630)) value at pH 7.4 in thefourth round and showing the largest difference between OD_((450/630))values at pH 7.4 and pH 6.0, with 8 gradients and 3-fold dilution, inwhich the abscissa represents the dilution factor, and the ordinaterepresents the OD value. The results show that the pH-dependent antigenbinding effect is better presented after the gradient dilution, in whichD3, D4 and D5 show the better performance, and D5 molecule has the besteffect (the remaining 5 molecules are not shown).

FIG. 5 shows a summary of the mutation sites of C26, C27, C32, D3, D4and D5.

FIG. 6A shows the detection results of binding to HBsAg at pH 7.4 and pH6.0 for the quantified cell supernatant obtained from the small scaleeukaryotic transfection of C32, C27 and C26 in Example 3. FIG. 6B showsthe detection results of binding to HBsAg at pH 7.4 and pH 6.0 for thequantified cell supernatant obtained from the small scale eukaryotictransfection of D3, D4 and D5 in Example 3. The abscissa represents theantibody concentration (Log 10 ng/ml), and the ordinate represents theOD value. The results show that C32, C27, C26, D3, D4 and D5 all canmaintain an antigen-binding activity equivalent to that of the parentantibody 162 at neutral pH, and all have a significant decrease inbinding activity to antigen at pH 6.0.

FIG. 7 shows the working principle of scavenger antibody. ThepH-dependent antigen binding activity plays a role in cells. Thus, ifthis first limiting factor of cell entry is not broken, the pH-dependentantigen-binding properties will not be applied subsequently, and thebenefit of modification will be greatly reduced. A scavenger antibodyobtained by further mutation of amino acids in the Fc region can enhancethe binding to hFcRn receptor at neutral pH, or enhance the binding toFcγRs receptor. Tthe scavenger antibody is located outside the cell andacts as a “transport helper” for reciprocally transporting antigens intothe cell, the antibody half-life can thus be extremely prolonged, and itcan bind to antigen again, thereby improving the cell entry efficiencyof antigens, and greatly improving the clearance efficiency.

FIGS. 8A to 8B show the protein gel results of pH-dependent antibodiesand antibodies with DY modification. FIG. 8A: the picture of protein gelof pH-dependent antibodies, in which 162 is a positive control, and theresults show that the expressed C26, D3, D4 and D5 antibodies aresingle-component. FIG. 8B: the picture of protein gel of antibodies withDY modification, in which 162 is a positive control, and the resultsshow that the expressed antibodies C26 DY, D3 DY, D4 DY and D5 DY aresingle-component.

FIGS. 9A to 9D show the detection results of pH-dependent antibodies andantibodies with DY modification binding to HBsAg at pH 7.4 and pH 6.0 inExample 4, in which the abscissa represents the antibody concentration(Log 10 ng/ml) and the ordinate represents the OD value. The resultsshow that D26, D3, D4 and D5 can maintain an antigen-binding activityequivalent to that of the parent (162) at the neutral pH, and have asignificant decrease in antigen-binding activity under the condition ofpH 6.0, and the corresponding antibodies with DY modification also canmaintain an antigen-binding activity at the neutral pH and apH-dependent antigen-binding activity comparable to those of the parent.

FIG. 10A shows the immunofluorescence experiment of mouse primarymacrophages for the pH-dependent antibodies and antibodies with DYmodification in Example 4, in which the green fluorescence representshFcRn, the blue fluorescence represents nucleus, and the redfluorescence represents HBsAg. The results show that the DY modificationenhances the phagocytosis of murine macrophages to the antigen-antibodycomplexes. FIG. 10B shows phagocytosis experiment based on human THP-1phagocytic cells of the pH-dependent antibodies and the antibodies withDY modification in Example 4. The results show that the DY modificationenhances the phagocytosis of human THP-1 phagocytic cells to theantigen-antibody complexes.

FIGS. 11A to 11B show the therapeutic effects of the C26 DY scavengerantibody and 162 in HBV transgenic mice after injection with a singledose of 5 mg/kg via tail vein in Example 4. FIG. 11C shows thetherapeutic effects of the D3 DY, D3 DY, D4 DY and D5 DY in HBVtransgenic mice after injection with a single dose of 5 mg/kg via tailvein in Example 4. FIG. 11A: the abscissa represents the number of days(d) after the injection of antibody, and the ordinate represents theHBsAg level in mouse serum after clearance (log 10 IU/ml). FIG. 11Bshows: the changes in the concentration of antibody in mouse serum, inwhich the abscissa represents the number of days (d) after the injectionof antibody, and the ordinate represents the antibody concentration(ng/ml). FIG. 11C: the abscissa represents the number of days (d) afterthe injection of antibody, and the ordinate represents the HBsAg levelin mouse serum after clearance (log 10 IU/ml). The results show that thescavenger antibodies with DY modification C26 DY, D3 DY, D4 DY and D5 DYare stronger in antigen clearance ability by more than one order ofmagnitude than 162. This indicates that the scavenger antibodies with DYmodification C26 DY, D3 DY, D4 DY and D5 DY could play the function ofcyclically binding antigens and enhance the effect of antigen clearanceat alow injection dose of 5 mg/kg.

SEQUENCE INFORMATION

Information of partial sequences involved in the present invention isprovided in Table 1 below.

TABLE 1 Description of sequences SEQ ID NO Description Sequence information  1 162 VHEVQLQESGPGLVKPSQTLSLTCAVSGSSITYGYHWNWIRQFPGNKLEWIGYISYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS  2C27 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNT HVPYTFGGGTKLEIK  3C26 D4 D5 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS  4C26 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHVVYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNT HHPYTFGGGTKLEIK  5C27 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYINHDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS  6C32 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITYRYHWNWIRQFPGNKLEWIGYINYDGSVHYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYY CASGFDHWGQGTTLTVSS  7C32 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHVVYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNT HLPYTFGGGTKLEIK  8D3 VH EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHYNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYC ASGFDHWGQGTTLTVSS  9D3 D5 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQN THVPYTFGGGTKLEIK 10D4 VK DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQN THLPYTFGGGTKLEIK 11162 YGYHWN HCDR1 12 162 D3 D4 D5 YISYDGSVLYNPSLEN HCDR2 13162 C26 C27 C32 GFDH D3 D4 D5 HCDR3 14 162 C26 C27 C32 RSSQSLVHSYGDTYLHLCDR1 15 162 C26 C27 C32 KVSNRFS D3 D4 D5 LCDR2 16 162 C27 D3 D5SQNTHVPYT LCDR3 17 C26 C27 D4 D5 HGYHWN HCDR1 18 C26 YIHYDGSVLYNPSLENHCDR2 19 C26 SQNTHHPYT LCDR3 20 C27 YINHDGSVQYNPSLEN HCDR2 21 C32 YRYHWNHCDR1 22 C32 YINYDGSVHYNPSLEN HCDR2 23 C32 D4 SQNTHLPYT LCDR3 24 D3HGYHYN HCDR1 25 D3 D4 D5 RSSQSLVHSYGDNYLH LCDR1 26 General formula ofX₁X₁YHX₁N HCDR1 27 General formula of YIX₄X₅DGSVX₆YNPSLEN HCDR2 28General formula of RSSQSLVHSYGDX₇YLH LCDR1 29 General formula ofSQNTHX₈PYT LCDR3 30 C26 C27 C32 D3 D4 EVQLQESGPGLVKPSQTLSLTCAVSGSSIT D5HFR1 31 C26 C27 C32 D3 D4 WIRQFPGNKLEWIG D5 HFR2 32 C26 C27 C32 D3 D4RVTITRDTSKNQFFLKLSSVTAEDTAKYYCAS D5 HFR3 33 C26 C27 C32 D3 D4WGQGTTLTVSS D5 HFR4 34 C26 C27 C32 D3 D4 DVVMTQSPLSLPVTLGEPASISC D5 LFR135 C26 C27 C32 D3 D4 WYLQKPGQSPKLLIY D5 LFR2 36 C26 C27 C32 D3 D4GVPDRFSGSGSGTDFTLKISRVETEDLGVYYC D5 LFR3 37 C26 C27 C32 D3 D4 FGGGTKLEIKD5 LFR4 38 4-28-02 QVQLQESGPGLVKPSQTLSLTCAVSGYSISSSNWWGW1RQPPGKGLEWIGYIYYSGSIYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAVDTAVYY CAR 39 2D-28-01DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 40 Human IgG1 heavyASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS chain constantGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK regionKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMTSRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 41 Human κ light chainRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDSAL constant regionQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 42Human IgG1 heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSchain constant GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKregion with V4 KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVmutation VVDVSHEDPEVKFNWYVDGVEVHEAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK 43 Human IgG1 heavyASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS chain constantGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK region with DYKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV mutationVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNDAYPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 44Primer 5′>GTTATTACTCGTGGCCCAGCCGGCCATGGCAGAGGTGCAGCTGC AGGAGTC <3′ 45Primer 5′>CTCCAGCTTGTTCCCTGGGAACTGCCGGATCCAGTTSYRGTGGTRGYSGTRGGTGATGGAGCTACCAGA <3′ 46 Primer5′>GTTCCCAGGGAACAAGCTGGAGTGGATTGGGYACMWCMRCYACSACGGCAGCSWYCWSYACAATCCATCTCTCG <3′ 47 Primer5′>GACTGTGAGAGTTGTGCCTTGGCCCCAGTGGTSGWRACCACTCG CACAGTA <3′ 48 Primer5′>CCAGATCCGCCACCTCCACTCCCGCCTCCACCTGAGGAGACTGT GAGAGTTGTGCCTT <3′ 49Primer 5′>GTGGAGGTGGCGGATCTGGAGGGGGTGGTAGCGATGTTGTGAT GACCCAATC <3′ 50Primer 5′>CTTTGGAGACTGGCCTGGCTTCTGCAGGTACCAATGSWGGTRGKKGTCTCCATAGYKGTGRWS <3′ 51 Primer5′>AGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAAC CGATTTTCTG <3′ 52 Primer5′>TTTCCAGCTTGGTCCCCCCTCCGAAGKKGTRGKGRWSATGGKKG TKSTGAGAGCAGTAATAAAC <3′53 Primer 5′>TAGTCGACCAGGCCCCCGAGGCCTTTTATTTCCAGCTTGGTCCC CCCT <3′ 54Signal peptide MGWSCIILFLVATATGVHS 55 Primer 5′-AGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGCAGGA GTC 56 Primer 5′-GATGGGCCCTTGGTCGACGCTGAAGAGACGGTGACGGTGG 57 Primer 5′-AGTAGCAACTGCAACCGGTGTACATTCTGACATACAGATGACGCA GTCTC 58 Primer 5′-ATGGTGCAGCCACCGTACGTTTGATTTCCACCTTGGTCC

EXAMPLES

The present invention will now be described with reference to thefollowing examples which are intended to illustrate the presentinvention rather than limit the present invention.

Unless otherwise specified, the molecular biology experimental methodsand immunoassay methods used in the present invention basically refer toJ. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory Press, 1989, and F M Ausubel et al.,Compiled Molecular Biology Experiment Guide, 3rd edition, John Wiley &Sons, Inc., 1995; the restriction enzymes were used in accordance withthe conditions recommended by the product manufacturer. Those skilled inthe art know that the examples describe the present invention by way ofexample, and are not intended to limit the scope of protection sought tobe protected by the present invention.

Example 1: Phage Screening of pH-Dependent Anti-HBsAg Antibodies 1.1Determination of Mutation Sites for pH-Dependent Antibody Modification

The anti-HBV humanized antibody 162 (detailed in Chinese patentapplication 201610879693.5) developed in the laboratory was used as theparent antibody, and its variable region were modified for pH-dependentantigen binding. As shown in FIG. 1, the modified 162 could maintain theantigen-binding activity under neutral conditions, but itsantigen-binding activity under acidic conditions was greatly reduced.The dissociated modified 162 could bind to intracellular FcRn so as toreturn to the plasma and bind to the antigen again, so that one moleculeof the modified 162 with pH-dependent antigen binding ability couldrepeatedly bind and neutralize a plurality of molecules of antigen.Histidine was protonated under acidic conditions and was a key aminoacid to bring the pH-dependent antigen binding properties. The 162 Fabhad an analyzed crystal structure, the analyzed crystal structure wasdocked by simulation with an antigen short peptide, and part of theresults was shown in FIG. 2, in which the blue structure represented theantigen short peptide, and the red structure represented part of thebinding region of 162 antibody. According to the docking results, atotal of 14 key amino acids for antigen and antibody binding were found.Considering that the simulated docking results had greater referencevalue, the amino acids on the interface and the amino acids on the bothsides were selected for mutation, and 26 sites were determined.

1.2 Construction of Phage Library of pH-Dependent scFv Antbodies Derivedfrom 162

Using the variable regions of the light and heavy chains of the 162antibody as a template, the determined sites in the antibody variableregion CDRs were mutated for pH-dependent modification, and the targetfragments were amplified according to the primers in Table 2 to obtainthe gene fragments coding the pH-dependent scFv antibodies derived from162. PCR conditions were: 95° C., 5 min; 95° C., 30 s; 57° C., 30 s; 72°C., 30 s; 72° C., 10 min; for 25 amplification cycles; SOE-PCR reactionconditions were: 95° C., 5 min; 95° C., 30 s; 57° C., 30 s; 72° C., 30s; 72° C., 10 min; for 5 amplification cycles. The amplified productswere analyzed by agarose gel electrophoresis, and the amplificationproducts were recovered/purified by using the DNA purification andrecovery kit (TianGen, DP214-03), thereby obtaining the gene fragmentsH-K encoding the humanized scFv antibodies derived from 162. Thestructure of scFv antibodies was: NH₂-VH-linker-VL-COOH, and the linkersequence could be (G₄S)₃. Each of the gene fragments H-K was digestedwith SfiI, and then ligated to the vector pCGMT (from Scripps, Makingchemistry selectable by linking it to infectivity) at a molar ratio of10:1 (gene fragment:vector). The ligation products were transformed intocompetent Escherichia coli ER2738 by electroporation (electroporationconditions: 25 μF, 2.5 KV, 200 Ω). The transformed Escherichia coli wasrecovered in SOC medium for 45 min, and then 200 μL of bacterialsolution was plated on LB plates (comprising 100 g/Lampicillin+tetracycline+2 g/mL glucose), and incubated by standing at37° C. overnight. All colonies on the plates were the libraries that themutation sites determined in the variable regions were randomly mutatedinto histidine, which were used for subsequent screening. Monoclonalcolonies were picked out from the plates and sequenced to ensure thecorrectness of the sequences of recombinant vectors encoding the scFvantibodies. The schematic diagram of the recombinant vector (pCGMT-scFv)encoding the scFv antibody was shown in FIG. 3.

TABLE 2 Mutation primers for pH-dependent scFv antibodies derived from162 Primer name Primer sequence VH-F SEQ ID NO: 44 HCDR1-R SEQ ID NO: 45HCDR2-F SEQ ID NO: 46 HCDR3-R SEQ ID NO: 47 VH-R SEQ ID NO: 48 VK-F SEQID NO: 49 KCDR1-R SEQ ID NO: 50 KCDR2-F SEQ ID NO: 51 KCDR3-R SEQ ID NO:52 VK-R SEQ ID NO: 53

1.3 Detection of Humanized scFv Antibodies

The library obtained in the previous step was screened for multiplerounds, and the positive monoclonal colonies obtained in the screeningwere cultured with 2×YT medium containing ampicillin (100 g/L) andglucose (2 g/mL) to reach an OD value of 0.6, and then added with M13KO7for auxiliary super-infection. After 2 h, 100 g/L kanamycin was addedand the super-infection was performed at 37° C. After 2 h, the culturewas centrifuged at 4000 rpm for 10 min, the supernatant was discarded,and the cell pellet was collected. The cell pellet was resuspended in amedium containing ampicillin and kanamycin (100 g/L), and cultured withshaking at 30° C. overnight. Subsequently, the culture was centrifugedat 12000 rpm for 10 min, the cells and supernatant were collected, andstored at 4° C. for testing.

An ELISA plate coated with HBsAg (200 ng/mL) antigen was used, and 100μL of the supernatant to be tested was added to each well, and incubatedat 37° C. for 1 h (two wells for each supernatant). Subsequently, theELISA plate was washed once with PBST, and then the two wells of eachsupernatant were added with 120 μL of PBS with pH 7.4 and pH 6.0respectively and incubated at 37° C. for 30 min. After washing with PBSTof corresponding pH for 5 times, 100 μL , of anti M13-HRP diluted at1:5000 was added, and incubated at 37° C. for 30 min. Subsequently, theELISA plate was washed 5 times with PBST, and the substrate TMB solutionwas added. After 15 minutes of color development, the color reaction wasterminated with H₂SO₄, and the reading was measured at OD450/630. Thedetection results of ELISA of the third round were shown in FIGS. 4A to4D. The results showed that the phages displaying these scFv antibodiesall had reactivity in ELISA detection and weakly bound to antigens at pH6.0; six strains of pH-dependent phage antibodies with good effects wereinitially obtained, named C-26, C-27, C-32, D3, D4 and D5, respectively.

Example 2: Preparation of pH-Dependent Anti-HBsAg Antibodies 2.1Construction of Recombinant Vector for Eukaryotic Expression

In the present invention, a large amount of antibody recombinationneeded to be carried out, so it was necessary to construct a set oflight and heavy chain vectors that can efficiently recombine antibodies.In the present invention, the existing eukaryotic expression vector pTT5in the laboratory was specially modified to construct a set of light andheavy chain recombinant vectors for double plasmid co-transfection.MGWSCIILFLVATATGVHS (SEQ ID NO: 54) was used as the signal peptide forthe light and heavy chains. The sequences encoding the constant regionsof the human antibody light and heavy chains were separately ligated tothe downstream of signal peptide to construct a set of eukaryoticexpression vectors pTT5-CH, pTT5-Cκ and pTT5-Cλ that facilitatedantibody recombination.

The six scFv antibodies obtained in 1.3 were used to amplify the lightand heavy chain variable region fragments with the primers in Table 3.The specific amplification reaction conditions were: 95° C., 5 min; 95°C., 30 s; 57° C., 30 s; 72° C., 30 s; 72° C., 10 min; for 25amplification cycles. And the amplification products were recovered fromthe gel.

The laboratory-made Gibson assembly solution was used to ligate theabove constructed eukaryotic expression vector with the recovered PCRproduct of antibody variable region gene (the primer carried a sequencehomologous to the vector) to obtain the recombinant vectors VH+pTT5-CH(comprising the CH shown in SEQ ID NO: 40) and VH+pTT5-Cκ (comprisingthe CL shown in SEQ ID NO: 41). The recombinant vector was transformedinto E. coli DH5α strain, plated on LB plate, and cultivated overnightin a 37° C. incubator. Monoclonal colonies were picked out from theplate and sequenced, and the sequencing results were subjected tosequence comparison using MEGA to confirm the correctness of its genes,and exclude the genes with wrong information.

TABLE 3 Primers for construction of eukaryotic expression vectors Primername Primer sequence VH-F SEQ ID NO: 55 VH-R SEQ ID NO: 56 VK-F SEQ IDNO: 57 VK-R SEQ ID NO: 58

2.2 Small- and Large-Scale Expression of Antibody Genes

The constructed recombinant vectors VH+pTT5-CH and VH+pTT5-Cκ wereco-transfected into HEK293 cells, and double plasmids for small-scaleexpression were co-transfected into a 24-well plate, 500 μL per well; ifthe cell supernatant of small-scale expression had antigenic activity,the transfection system was enlarged to 100 mL (determined by the amountof antibody used) of FreeStyle™ 293F suspension cells (the cell densitywas about 2×10⁶ cells/ml). The transfected cells were cultured in ashake flask in a 32° C., 5% CO₂ incubator, and the supernatant wascollected after 7 days of expression.

2.3 Antibody Purification

The cell expression supernatant was collected and purified with aProtein A column according to the manufacturer's instructions. Thespecific steps were as follows: the harvested cell culture supernatantwas centrifuged at 8000 rpm for 10 min, the supernatant was retained,the pH value was adjusted to 8.4 with dry powder Na₂HPO₄, and thenfiltered with a filter membrane with 0.22 μm pore diameter. 10 mL ofSepharose 4B medium coupled with Protein A was loaded into column, itwas connected to AKTA Explorer100 system, the pump A was connected to0.2 M disodium hydrogen phosphate solution, and the pump B was connectedto 0.2 M citric acid solution. Detection wavelength was UV 280 nm, flowrate was 5 mL/min, and the sample injection proportion of pumps A/B wasadjusted. The column was first washed with 100% B (pH 2.3) to removeprotein impurities, the pH was balanced with 10% B (pH 8.0), the signalat the detection wavelength returned to zero after it was stable, thenthe sample was loaded. After the flow through peak passed, 10% B wasused for balance until the signal at the detection wavelength wasreduced to zero and was stable, elution was performed using 70% B (pH4.0), and the elution peak was collected. The elution peak sample wasdialyzed into PBS buffer and subjected to assay of concentration andSDS-PAGE and HPLC analysis to determine the purity of IgG antibody.

Example 3: Property Analysis and Functional Evaluation of pH-DependentAnti-HB sAg Antibodies

Through the method of Example 1, six strains of pH-dependent phageantibodies that bound to HBsAg were obtained by preliminary screening,named C26, C27, C32, D3, D4 and D5, respectively. Furthermore, thesmall-scale eukaryotic expression and purification of the 6 strains ofphage antibodies were carried out by the method of Example 2. The VH andVL amino acid sequences of the 6 antibodies were shown in Table 4 below.In addition, the CDR sequences of the 6 antibodies were determined, andthe CDR amino acid sequences of the heavy chain variable regions and thelight chain variable regions thereof were shown in Table 5. The mutationsites that endowed C26, C27, C32, D3, D4 and D5 with pH-dependentantigen binding properties to HBsAg were summarized in FIG. 5.

TABLE 4 Amino acid sequences of C26/C27/C32/D3/D4/D5 light and heavy chain variable regions Sequence  SEQ  name ID NOAmino acid sequence  C26 VH 3EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK YYCASGFDHWGQGTTLTVSSC26 VK 4 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHHYTEGGGTKLEIKC27 VH 5 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYINHDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK YYCASGFDHWGQGTTLTVSSC27 VK 2 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHVPYTFGGGTKLEIKC32 VH 6 EVQLQESGPGLVKPSQTLSLTCAVSGSSITYRYHWNWIRQFPGNKLEWIGYINYDGSVHYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK YYCASGFDHWGQGTTLTVSSC32 VK 7 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHLPYTFGGGTKLEIKD3 VH 8 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHYNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKY YCASGFDHWGQGTTLTVSSD3 VK 9 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHVPYTFGGGTKLEIKD4 VH 3 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK YYCASGFDHWGQGTTLTVSSD4 VK 10 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHLPYTFGGGTKLEIKD5 VH 3 EVQLQESGPGLVKPSQTLSLTCAVSGSSITHGYHWNWIRQFPGNKLEWIGYIHYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAK YYCASGFDHWGQGTTLTVSSD5 VK 9 DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDNYLHWYLQKPGQSPKLLIYKVSNRESGVPDRFSGSGSGTDFTLKISRVETEDLGVYYC SQNTHVPYTFGGGTKLEIK

TABLE 5 CDR sequences of C26/C27/C32/D3/D4/D5  light and heavy chainsC26 VH CDR1 HGYHWN SEQ ID NO: 17 VH CDR2 YIHYDGSVLYNPSLEN SEQ ID NO: 18VH CDR3 GFDH SEQ ID NO: 13 VL CDR1 RSSQSLVHSYGDTYLH SEQ ID NO: 14VL CDR2 KVSNRFS SEQ ID NO: 15 VL CDR3 SQNTHHPYT SEQ ID NO: 19 C27VH CDR1 HGYHWN SEQ ID NO: 17 VH CDR2 YINHDGSVQYNPSLEN SEQ ID NO: 20VH CDR3 GFDH SEQ ID NO: 13 VL CDR1 RSSQSLVHSYGDTYLH SEQ ID NO: 14VL CDR2 KVSNRFS SEQ ID NO: 15 VL CDR3 SQNTHVPYT SEQ ID NO: 16 C32VH CDR1 YRYHWN SEQ ID NO: 21 VH CDR2 YINYDGSVHYNPSLEN SEQ ID NO: 22VH CDR3 GFDH SEQ ID NO: 13 VL CDR1 RSSQSLVHSYGDTYLH SEQ ID NO: 14VL CDR2 KVSNRFS SEQ ID NO: 15 VL CDR3 SQNTHLPYT SEQ ID NO: 23 D3 VH CDR1HGYHYN SEQ ID NO:24 VH CDR2 YISYDGSVLYNPSLEN SEQ ID NO:12 VH CDR3 GFDHSEQ ID NO:13 VL CDR1 RSSQSLVHSYGDNYLH SEQ ID NO:25 VL CDR2 KVSNRFSSEQ ID NO: 15 VL CDR3 SQNTHVPYT SEQ ID NO:16 D4 VH CDR1 HGYHWNSEQ ID NO:17 VH CDR2 YISYDGSVLYNPSLEN SEQ ID NO:12 VH CDR3 GFDHSEQ ID NO:13 VL CDR1 RSSQSLVHSYGDNYLH SEQ ID NO:25 VL CDR2 KVSNRFSSEQ ID NO: 15 VL CDR3 SQNTHLPYT SEQ ID NO:23 D5 VH CDR1 HGYHWNSEQ ID NO:17 VH CDR2 YISYDGSVLYNPSLEN SEQ ID NO:12 VH CDR3 GFDHSEQ ID NO:13 VL CDR1 RSSQSLVHSYGDNYLH SEQ ID NO:25 VL CDR2 KVSNRFSSEQ ID NO:15 VL CDR3 SQNTHVPYT SEQ ID NO:16

The inventors first performed small-scale transfection for the sixmonoclonal antibodies; after quantifying the supernatant aftertransfection, the pH-dependent antigen binding ability with HBsAg wasdetected by ELISA method, and the antibody concentration was uniformlydiluted to 1111.11 ng/mL. Subsequently, 20% NBS was used to carry out a3-fold concentration gradient dilution of the antibody concentration,for a total of 8 concentration gradients. Subsequently, the dilutedantibody was added to a commercial HBsAg plate (purchased from BeijingWantai), and incubated at 37° C. for 1 h (two wells per supernatant).Subsequently, the ELISA plate was washed once with PBST and spin-dried.Then, the two wells of each supernatant were added with 120 μL, of pH7.4 and pH 6.0 PBS respectively and incubated at 37° C. for 30 min. Itwas then washed 5 times with PBST of corresponding pH and spin-dried.Subsequently, GAH-HRP-labeled secondary antibody was added, incubatedfor 30 min, the plate was washed 5 times with PBST, and spin-dried. Thesubstrate TMB solution was added. After 15 minutes of color development,the color reaction was terminated with H₂SO₄, and the reading wasmeasured at OD450/630.

The results were shown in FIGS. 6A to 6B. It could be seen from theresults that the candidate molecules all could maintain anantigen-binding activity comparable to that of the parent antibody 162at the neutral pH, and the antigen-binding activity was significantlyreduced at pH 6.0. The EC50 results were summarized in Table 6.

TABLE 6 EC50 values of pH-dependent activity detection for C26, C27,C32, D3, D4 and D5 EC50 in pH 6.0 EC50 in pH 7.4 EC50(pH 6.0)/ antibody(ng/mL) (ng/mL) EC50(pH 7.4) C26 331.30 37.20 8.90 C27 949.60 491.301.93 C32 2255.00 1333 1.69 D3 473.10 35.43 13.35 D4 188.10 37.16 5.06 D560.04 21.04 2.85

Example 4: Construction and Functional Evaluation of Scavenger Antibody

The pH-dependent antibody needs to enter the cell to exert itspH-dependent antigen-binding activity. Therefore, if the first limitingfactor of cell entry is not broken, the subsequent pH-dependentantigen-binding properties will have no chance to “play”. Therefore, inthis example, the scavenger antibody was obtained by further mutation ofamino acids in the Fc region, which could enhance the binding to hFcRnreceptor at neutral pH, or enhance the binding to FcγRs receptor. Asshown in FIG. 7, the scavenger antibody is located outside the cell andplayed the role of a “transportation helper” that reciprocallytransported antigens into the cell, thereby extremely extending theantibody half-life, and it could bind to antigen again, therebyimproving the efficiency of cell entry of antigen, and significantlyimproving the clearance efficiency.

The C26, D3, D4 and D5 were selected as the antibodies for subsequentevaluation, and subjected to Fc DY (K326D, L328Y) mutations to enhancethe affinity with mFcγRII under neutral conditions (the modification ofC26 Fc was commissioned to the General Biologicals, order numberG122413) to obtain scavenger antibodies C26 DY, D3 DY, D4 DY and D5 DYthat bound to mFcγRII. The above antibodies were subjected tolarge-scale eukaryotic expression and purification, and the specificsteps were same as Examples 1.2 and 1.3.

FIGS. 8A to 8B showed the protein gel results of the original antibodyand the modified antibodies. FIG. 8A: the picture of protein gel of theoriginal antibody, in which the 162 was a positive control. The resultsshowed that the expressed original antibody is single-component. FIG.8B: the picture of protein gel of antibodies with DY modification, inwhich the 162 was a positive control. The results showed that theexpressed antibodies with DY modification are single-component.

4.1 Evaluation of pH-Dependent Antigen-Binding Activity of ScavengerAntibodies Binding to mFcγRII

For the original antibody and the antibodies with DY modification afterexpression and purification, the inventors used the ELISA method todetect their pH-dependent antigen binding ability to HBsAg. First, a BCAprotein quantification kit was used to determine the concentrations ofthe purified antibodies, and the antibodies were uniformly diluted tohave a concentration of 1111.11 ng/mL. Subsequently, 20% NBS was used tocarry out a 3-fold concentration gradient dilution for the antibodyconcentrations, for a total of 8 concentration gradients. Subsequently,the diluted antibody was added to a commercial HBsAg plate (purchasedfrom Beijing Wantai) and incubated at 37° C. for 1 h (two wells persupernatant). Subsequently, the ELISA plate was washed once with PBSTand spin-dried. Then the two wells of each supernatant were added with120 μL of pH 7.4 PBS and pH 6.0 PBS respectively incubated at 37° C. for30 min, washed 5 times with PBST of corresponding pH and spin-dried.Subsequently, GAH-HRP-labeled secondary antibody was added, andincubated for 30 min, the plate was washed 5 times with PBST, andspin-dried. And the substrate TMB solution was added. After 15 minutesof color development, the color reaction was terminated with H₂SO₄, andthe reading was measured at OD450/630.

The results were shown in FIGS. 9A to 9D, in which the C26, D3, D4, D5and their DY modification antibodies all had an antigen-binding activityequivalent to that of antibody 162, but showed a weak binding to antigenat pH 6.0, thereby exhibiting a good pH-dependent antigen-bindingactivity.

4.2 Verification of Function at Cellular Level for Scavenger AntibodiesBinding mFcγRII 4.2.1 Labeling HBsAg with 488 Fluorescence

Take the labeling of 1 mg HBsAg as an example, the whole process wasprotected from light.

(1) 1 mL of 1 mg/mL HBsAg was dialyzed into borate buffer (PH 8.5, 500mL), 4° C., 4 h;

(2) the molar ratio of HBsAg to 488 label was 1:5, and 0.1988 mg of 488fluorescence was required after calculation;

(3) 10 mg/mL of 488 fluorescence solution was prepared with DMF andmixed well;

(4) 19.88 pL of 488 fluorescence was added to 1 mL of the dialyzedHBsAg, mixed well, and incubated at room temperature for 1 h;

(5) the incubation mixture was dialyzed into PBS at 4° C. overnight.

4.2.2 Immunofluorescence Experiment Based on Mouse Primary Macrophages

(1) 4 days before the experiment, 1.5 mL of 3% sodium thioglycolatesolution was injected into the abdominal cavity of each mouse, withoutinjecting into the intestine;

(2) two mice were executed and soaked in 75% alcohol for 3 minutes;

(3) the mouse was horizontally fixed on a foam board to expose theabdomen; the abdominal skin was cut with tissue scissors, the peritoneumwas disinfected and incised to expose the abdominal cavity, theabdominal incision skin was pulled by two toothed forceps hold in theleft hand and fixed, 1640 culture medium was pipetted by Pasteur pipettehold in the right hand for peritoneal lavage with 4 mL/time, for a totalof two times. The pipette was used to gently and fully stir theabdominal cavity to make the lavage more fully and thoroughly. Afterfully stirring for about 2 minutes and standing for about 5 minutes tofully isolate the macrophages, the lavage solution was pipetted andtransferred into a centrifuge tube;

(4) 4° C., 1100 g, 5 min;

(5) the supernatant was carefully discard, the cells were washed twicewith 1640 medium, 4° C., 1100 g, 5 min, the supernatant was discarded,and the cells were resuspended in RPM1640;

(6) After counting the cells, the cell density was adjusted to 10⁶cells/mL, the cells were cultured on a 24-well glass-bottom cell imagingculture plate, 250 μL/well, the medium was replaced after 2 h, andwashing was carried out once with RPM1640, after the non-adherent cellswere discarded, the cells were incubated overnight in a 37° C., CO₂incubator;

(7) the antibody and antigen labeled with the corresponding fluorescencewere diluted in serum-free medium to: 800 ng/mL for antigen and 20 μg/mLfor antibody;

(8) 125 μL of the antigen and 125 μL of the antibody were mixeduniformly, and then were allowed to stand for 1 hour in a 37° C., CO₂incubator;

(9) the cell supernatant in the cell imaging culture plate wasdiscarded, the antigen-antibody complex was added, shaken evenly, andallowed to stand in a 37° C., CO₂ incubator for 2 hours;

(10) the supernatant was discarded, and 1 mL of sterile PBS incubated at37° C. in advance was used to “wash” the cell surface 3 to 5 times, andthen totally removed by a pipette;

(11) the 1:2000 diluted Dio was added in an amount that immersed thecells, and allowed to stand at room temperature for 20 min;

(12) the supernatant was discarded, and 1 mL of sterile PBS incubated at37° C. in advance was used to “wash” the cell surface 3 to 5 times, andthen totally removed by a pipette;

(13) a live cell nuclear dye was added (2 drops were added to 1 mL ofvolume), allowed to stand at room temperature for 20 min, and placed ina high-content imager for imaging.

The results of the experiment were shown in FIG. 10A. It could be seenfrom the results that the DY modification enhanced the phagocytosis ofmouse macrophages to the antigen-antibody complexes, leading to moreantigen degradation.

4.2.3 Validation by Chemiluminescence Method Based on human THP-1Phagocytic Cells

(1) Adherent THP-1 cells were coated on a plate at 2×10⁵/well, addedwith 1640 medium containing 10% serum, placed in a carbon dioxideincubator and cultured at 37° C. for 24 h;

(2) HBsAg was diluted with serum-free 1640 medium to 800 ng/mL, and theantibody to be tested with 20 ug/mL as the initial concentration wassubjected to 2-fold gradient dilution, for a total of 11 gradients. 300uL of the diluted HBsAg and 300 uL of the antibody to be tested weremixed at ratio of 1:1, and allowed to stand at 37° C. for 1 h;

(3) the THP-1 cell supernatant was discarded, 250 uL of theHBsAg-antibody mixture was added to the THP-1 cells, placed in a carbondioxide incubator and cultured at 37° C. for 1 hour;

(4) the THP-1 cell supernatant was discarded, and washed 3 times withsterile PBS;

(5) 120 uL of DDM cell lysis solution was added to each well of THP-1cells and allowed to stand and react at 4° C. for 1 hour;

(6) the supernatant of the lysate was subjected to detecting theconcentration of HBsAg by using hepatitis B surface antigen quantitativedetection kit (Beijing Wantai).

The results of the experiment were shown in FIG. 10B. It could be seenfrom the results that the DY modification enhanced the phagocytosis ofhuman THP-1 phagocytic cells to the antigen-antibody complexes.

4.3 Determination of Therapeutic Effect of Scavenger Antibody Binding tomFcγRI in Animal Models

HBV transgenic mice were selected as animal models. The C26 DY, D3 DY,D4 DY and D5 DY scavenger antibodies and 162 were injected at a singledose of 5 mg/kg via tail vein (4 mice in each group) to the 6-8 weeksold HBV transgenic mice. By detecting the concentrations of HBsAg,antibody and HBV DNA in serum, the antigen clearance rates and antibodyhalf-life of the scavenger antibodies in vivo were analyzed.

Quantitative Detection of HBsAg

(1) Preparation of reaction plate: the mouse monoclonal antibodyHBs-45E9 was diluted with 20 mM PB buffer (Na₂HPO₄/NaH₂PO₄ buffer, pH7.4) to 2 μg/mL, and 100 μL of coating solution was added to each wellof a chemiluminescence plate, and the coating was carried out at 2-8° C.for 16-24 h, followed by another 2 hours at 37° C., the plate was washedonce with PBST washing solution, and spin-dried. After washing, 200 μLof blocking solution was added to each well and the blocking was carriedout at 37° C. for 2 h. Subsequently, the blocking solution wasdiscarded, and the plate was placed in a drying room to dry, and storedat 2-8° C. for later use.

(2) Sample dilution: the collected mouse serum was diluted with a PBSsolution containing 20% NB S (newborn bovine serum) at two gradients of1:30 and 1:150 for subsequent quantitative detection.

(3) Sample denaturation treatment: 15 μL of the above-diluted serumsample was mixed well with 7.5 μL of denaturation buffer (15% SDS,dissolved in 20 mM PB7.4), and reacted at 37° C. for 1 h. Then, 90 μL ofstop buffer (4% CHAPS, dissolved in 20 mM PB7.4) was added, and mixedwell.

(4) Sample reaction: 100 μL of the above-mentioned denatured serumsample was added to a reaction plate, and reacted at 37° C. for 1 hour.Subsequently, the reaction plate was washed 5 times with PBST andspin-dried.

(5) Enzymatic label reaction: the HBs-A6A7-HRP reaction solution wasadded at 100 μL/well to a chemiluminescence plate, and reacted at 37° C.for 1 h. Then, the plate was washed 5 times with PBST and spin-dried.

(6) Luminescence reaction and measurement: a luminescence solution (100μL/well) was added to the chemiluminescence plate, and light intensitymeasurement was performed.

(7) Calculation of HBsAg concentration in mouse serum sample: parallelexperiments were performed using standard products, and a standard curvewas drawn based on the measurement results of the standard products.Then, the light intensity measurement value of the mouse serum samplewas substituted into the standard curve, and the concentration of HBsAgin the serum sample to be tested was calculated.

The results of the detection of HBsAg in the serum were shown in FIG.11A and FIG. 11C. It could be seen from FIG. 11A that the scavengerantibody with DY modification C26 DY had an antibody clearance abilitystronger more than one order of magnitude than that of 162, which wasconsistent with the detection results of antibody half-life in the serum(FIG. 11B). In the comparison of the concentrations of antibodies in theserum, the half-life of C26 DY was longer than that of 162 by nearly 12days, which indicated that the scavenger antibody C26 DY had thefunction of circularly and reciprocally binding antigen, therebyincreasing the duration time of antigen clearance. The experimentalresults in FIG. 11C showed that the antigen clearance ability of D3 DY,D4 DY and D5 DY was equivalent to that of C26 DY, and the duration timewas longer than that of C26 DY, indicating that at a low injection doseof 5 mg/kg, the scavenger antibodies with DY modification D3 DY, D4 DYand D5 DY had a better function of circularly and reciprocally bindingantigen, thereby performing better antigen clearance.

Example 5: Affinity Determination of 162 and C26

HBsAg was dissolved in sodium acetate (pH 4.5) at 5 μg/mL, and the chipcoating program was run on the Biacore 3000 device to coat HBsAg on theCM5 chip. The coating volume of HBsAg was 2400 RU. The analyte wasdiluted 2-fold from 100 nM to prepare samples of 7 concentrations. Theaffinity determination program was run on the Biacore 3000 device, theflow rate was set to 50 μl/min, the binding time was set to 90 s, thedissociation time was set to 600 s, the temperature of sample chamberwas set to 10° C., the regeneration solution was 50 mM NaOH, theregeneration flow rate was set to 50 μL/min, and the regeneration timewas set to 60 s. The results were summarized in Table 7.

TABLE 7 Affinity determination of 162 and C26 KD(M) in KD(M) in KD(pH6.0)/ Antibody pH 7.4 pH 6.0 KD(pH 7.4) 162 9.34E−10 C26 3.45E−099.82E−09 2.85

Although the specific embodiments of the present invention have beendescribed in details, those skilled in the art will understand thatvarious modifications and changes can be made to the details accordingto all the teachings that have been published, and these changes arewithin the protection scope of the present invention. All of the presentinvention is given by the appended claims and any equivalents thereof.

What is claimed is:
 1. An antibody or antigen-binding fragment thereofcapable of specifically binding to HBsAg, wherein the antibody orantigen-binding fragment thereof binds to HBsAg with higher affinity atneutral pH than at acidic pH, and the antibody or antigen-bindingfragment thereof comprises: (a) a heavy chain variable region (VH)comprising the following 3 CDRs: (i) HCDR1 with a sequence of X₁X₂YHX₃N(SEQ ID NO: 26), wherein X₁ is selected from Y or H, X₂ is selected fromG or R, X₃ is selected from W or Y; (ii) HCDR2 with a sequence ofYIX₄X₅DGSVX₆YNPSLEN (SEQ ID NO: 27), wherein X₄ is selected from S, N orH, X₅ is selected from Y or H, X₆ is selected from L, H or Q; and (iii)HCDR3 with a sequence of GFDH (SEQ ID NO: 13); and/or, (b) a light chainvariable region (VL) comprising the following 3 CDRs: (iv) LCDR1 with asequence of RSSQSLVHSYGDX₇YLH (SEQ ID NO: 28), wherein X₇ is selectedfrom T or N; (v) LCDR2 with a sequence of KVSNRFS (SEQ ID NO: 15); and(vi) LCDR3 with a sequence of SQNTHX₈PYT (SEQ ID NO: 29), wherein X₈ isselected from V, L or H.
 2. The antibody or antigen-binding fragmentthereof according to claim 1, wherein the antibody or antigen-bindingfragment thereof comprises: (a) a heavy chain variable region (VH)comprising the following 3 CDRs: (i) HCDR1, consisting of a sequenceselected from the following: SEQ ID NOs: 17, 21, 24; (ii) HCDR2,consisting of a sequence selected from: SEQ ID NOs: 12, 18, 20, 22; and(iii) HCDR3, consisting of a sequence shown in SEQ ID NO: 13; and/or,(b) a light chain variable region (VL) comprising the following 3 CDRs:(iv) LCDR1, consisting of a sequence selected from the following: SEQ IDNOs: 14, 25; (v) LCDR2, consisting of a sequence shown in SEQ ID NO: 15;and (vi) LCDR3, consisting of a sequence selected from the following:SEQ ID NOs: 16, 19,
 23. 3. The antibody or antigen-binding fragmentthereof according to claim 1 or 2, wherein the antibody orantigen-binding fragment thereof comprises: (1) a VH comprising thefollowing 3 CDRs: HCDR1 shown in SEQ ID NO: 21, HCDR2 shown in SEQ IDNO: 22, HCDR3 shown in SEQ ID NO: 13; and, a VL comprising the following3 CDRs: LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15,LCDR3 shown in SEQ ID NO: 23; (2) a VH comprising the following 3 CDRs:HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, HCDR3 shownin SEQ ID NO: 13; and, a VL comprising the following 3 CDRs: LCDR1 shownin SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ IDNO: 19; (3) a VH comprising the following 3 CDRs: HCDR1 shown in SEQ IDNO: 17, HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 13; and,a VL comprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 14,LCDR2 shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 16; (4) a VHcomprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 24, HCDR2shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 13; and, a VLcomprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 25, LCDR2shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 16; (5) a VHcomprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 17, HCDR2shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 13; and, a VLcomprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 25, LCDR2shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO: 23; or (6) a VHcomprising the following 3 CDRs: HCDR1 shown in SEQ ID NO: 17, HCDR2shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 13; and, a VLcomprising the following 3 CDRs: LCDR1 shown in SEQ ID NO: 25, LCDR2shown in SEQ ID NO: 15, LCDR3 shown in SEQ ID NO:
 16. 4. The antibody orantigen-binding fragment thereof according to any one of claims 1 to 3,wherein the antibody or antigen-binding fragment thereof furthercomprises a framework region of a human immunoglobulin (for example, aframework region contained in an amino acid sequence encoded by a humangermline antibody gene), and the framework region optionally comprisesone or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) backmutations from human residues to murine residues; preferably, theantibody or antigen-binding fragment thereof comprises: a heavy chainframework region contained in an amino acid sequence encoded by a humanheavy chain germline gene, and/or a light chain framework regioncontained in an amino acid sequence encoded by a human light chaingermline gene; preferably, the antibody or antigen-binding fragmentthereof comprises: a heavy chain framework region contained in an aminoacid sequence encoded by human heavy chain germline gene 4-28-02 (SEQ IDNO: 38), and a light chain framework region contained in an amino acidsequence encoded by human light chain germline gene 2D-28-01 (SEQ ID NO:39), and the heavy chain framework region and/or the light chainframework region optionally comprises one or more (for example, 1, 2, 3,4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to murineresidues; preferably, the VH of the antibody or antigen-binding fragmentthereof comprises: VH FR1 as shown in SEQ ID NO: 30, VH FR2 as shown inSEQ ID NO: 31, VH FR3 as shown in SEQ ID NO: 32, and VH FR4 shown in SEQID NO: 33; preferably, the VL of the antibody or antigen-bindingfragment thereof comprises: VL FR1 as shown in SEQ ID NO: 34, VL FR2 asshown in SEQ ID NO: 35, VL FR3 as shown in SEQ ID NO: 36, and VL FR4shown in SEQ ID NO:
 37. 5. The antibody or antigen-binding fragmentthereof according to any one of claims 1 to 4, wherein the antibody orantigen-binding fragment thereof comprises: (a) a heavy chain variableregion (VH), which comprises an amino acid sequence selected from thefollowing: (i) a sequence shown in any one of SEQ ID NOs: 3, 5, 6, 8;(ii) a sequence with substitution, deletion or addition of one orseveral amino acids (for example, substitution, deletion or addition of1, 2, 3, 4 or 5 amino acids) as compared with a sequence shown in anyone of SEQ ID NOs: 3, 5, 6, 8; or (iii) a sequence with a sequenceidentity of at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% as compared with asequence shown in any one of SEQ ID NOs: 3, 5, 6, 8; and (b) a lightchain variable region (VL), which comprises an amino acid sequenceselected from the following: (iv) a sequence shown in any one of SEQ IDNOs: 2, 4, 7, 9, 10; (v) a sequence with substitution, deletion oraddition of one or several amino acids (for example, substitution,deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with asequence shown in any one of SEQ ID NOs: 2, 4, 7, 9, 10; or (vi) asequence with a sequence identity of at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% as compared with a sequence shown in any one of SEQ ID NOs: 2, 4,7, 9, 10; preferably, the substitution described in (ii) or (v) is aconservative substitution.
 6. The antibody or antigen-binding fragmentthereof according to any one of claims 1 to 5, wherein the antibody orantigen-binding fragment thereof comprises: (1) a VH with a sequenceshown in SEQ ID NO: 3 and a VL with a sequence shown in SEQ ID NO: 4;(2) a VH with a sequence shown in SEQ ID NO: 5 and a VL with a sequenceshown in SEQ ID NO: 2; (3) a VH with a sequence shown in SEQ ID NO: 6and a VL with a sequence shown in SEQ ID NO: 7; (4) a VH with a sequenceshown in SEQ ID NO: 8 and a VL with a sequence shown in SEQ ID NO: 9;(5) a VH with a sequence shown in SEQ ID NO: 3 and a VL with a sequenceshown in SEQ ID NO: 10; or (6) a VH with a sequence shown in SEQ ID NO:3 and a VL with a sequence shown in SEQ ID NO:
 9. 7. The antibody orantigen-binding fragment thereof according to any one of claims 1 to 6,wherein the antibody or antigen-binding fragment thereof furthercomprises a constant region derived from a human immunoglobulin;preferably, the heavy chain of the antibody or antigen-binding fragmentthereof comprises a heavy chain constant region derived from a humanimmunoglobulin (for example, IgG1, IgG2, IgG3 or IgG4), and the lightchain of the antibody or antigen-binding fragment thereof comprises alight chain constant region derived from a human immunoglobulin (forexample, κ or λ); preferably, the antibody or antigen-binding fragmentthereof comprises a light chain constant region (CL) as shown in SEQ IDNO:
 41. 8. The antibody or antigen-binding fragment thereof according toany one of claims 1 to 7, wherein the antibody or antigen-bindingfragment thereof comprises a variant of a human IgG1 heavy chainconstant region, the variant has the following substitution as comparedto a wild-type sequence from which it is derived: (i) M252Y, N286E,N434Y; or, (ii) K326D, L328Y; wherein the above-mentioned amino acidpositions are positions according to the Kabat numbering system;preferably, the antibody or antigen-binding fragment thereof comprises aheavy chain constant region (CH) as shown in SEQ ID NO: 42 or
 43. 9. Theantibody or antigen-binding fragment thereof according to any one ofclaims 1 to 7, wherein the antibody or antigen-binding fragment thereofcomprises a heavy chain constant region (CH) as shown in SEQ ID NO: 40.10. The antibody or antigen-binding fragment thereof according to anyone of claims 1 to 9, wherein the antibody or antigen-binding fragmentthereof comprises: (1) a heavy chain comprising a VH shown in SEQ ID NO:3 and a CH shown in SEQ ID NO: 40, and a light chain comprising a VLshown in SEQ ID NO: 4 and a CL shown in SEQ ID NO: 41; (2) a heavy chaincomprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO: 47,and a light chain comprising a VL shown in SEQ ID NO: 4 and a CL shownin SEQ ID NO: 41; (3) a heavy chain comprising a VH shown in SEQ ID NO:3 and a CH shown in SEQ ID NO: 48, and a light chain comprising a VLshown in SEQ ID NO: 4 and a CL shown in SEQ ID NO: 41; (4) a heavy chaincomprising a VH shown in SEQ ID NO: 5 and a CH shown in SEQ ID NO: 40,and a light chain comprising a VL shown in SEQ ID NO: 2 and a CL shownin SEQ ID NO: 41; (5) a heavy chain comprising a VH shown in SEQ ID NO:5 and a CH shown in SEQ ID NO: 47, and a light chain comprising a VLshown in SEQ ID NO: 2 and a CL shown in SEQ ID NO: 41; (6) a heavy chaincomprising a VH shown in SEQ ID NO: 5 and a CH shown in SEQ ID NO: 48,and a light chain comprising a VL shown in SEQ ID NO: 2 and a CL shownin SEQ ID NO: 41; (7) a heavy chain comprising a VH shown in SEQ ID NO:6 and a CH shown in SEQ ID NO: 40, and a light chain comprising a VLshown in SEQ ID NO: 7 and a CL shown in SEQ ID NO: 41; (8) a heavy chaincomprising a VH shown in SEQ ID NO: 6 and a CH shown in SEQ ID NO: 47,and a light chain comprising a VL shown in SEQ ID NO: 7 and a CL shownin SEQ ID NO: 41; (9) a heavy chain comprising a VH shown in SEQ ID NO:6 and a CH shown in SEQ ID NO: 48, and a light chain comprising a VLshown in SEQ ID NO: 7 and a CL shown in SEQ ID NO: 41; (10) a heavychain comprising a VH shown in SEQ ID NO: 8 and a CH shown in SEQ ID NO:40, and a light chain comprising a VL shown in SEQ ID NO: 9 and a CLshown in SEQ ID NO: 41; (11) a heavy chain comprising a VH shown in SEQID NO: 8 and a CH shown in SEQ ID NO: 47, and a light chain comprising aVL shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41; (12) a heavychain comprising a VH shown in SEQ ID NO: 8 and a CH shown in SEQ ID NO:48, and a light chain comprising a VL shown in SEQ ID NO: 9 and a CLshown in SEQ ID NO: 41; (13) a heavy chain comprising a VH shown in SEQID NO: 3 and a CH shown in SEQ ID NO: 40, and a light chain comprising aVL shown in SEQ ID NO: 10 and a CL shown in SEQ ID NO: 41; (14) a heavychain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO:47, and a light chain comprising a VL shown in SEQ ID NO: 10 and a CLshown in SEQ ID NO: 41; (15) a heavy chain comprising a VH shown in SEQID NO: 3 and a CH shown in SEQ ID NO: 48, and a light chain comprising aVL shown in SEQ ID NO: 10 and a CL shown in SEQ ID NO: 41; (16) a heavychain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQ ID NO:40, and a light chain comprising a VL shown in SEQ ID NO: 9 and a CLshown in SEQ ID NO: 41; (17) a heavy chain comprising a VH shown in SEQID NO: 3 and a CH shown in SEQ ID NO: 47, and a light chain comprising aVL shown in SEQ ID NO: 9 and a CL shown in SEQ ID NO: 41; or (18) aheavy chain comprising a VH shown in SEQ ID NO: 3 and a CH shown in SEQID NO: 48, and a light chain comprising a VL shown in SEQ ID NO: 9 and aCL shown in SEQ ID NO:
 41. 11. The antibody or antigen-binding fragmentthereof according to any one of claims 1 to 10, wherein the antibody orantigen-binding fragment thereof is selected from the group consistingof scFv, Fab, Fab′, (Fab′)₂, Fv fragment, diabody, bispecific antibody,multispecific antibody, probody, chimeric antibody or humanizedantibody; preferably, the antibody is a chimeric antibody or a humanizedantibody.
 12. The antibody or antigen-binding fragment thereof accordingto any one of claims 1 to 10, wherein the antibody or antigen-bindingfragment thereof is able to specifically bind to HBsAg, neutralize avirulence of HBV, and/or reduce a serum level of HBV DNA and/or HBsAg ina subject.
 13. An isolated nucleic acid molecule, which encodes theantibody or antigen-binding fragment thereof according to any one ofclaims 1 to 12, or its heavy chain variable region and/or light chainvariable region.
 14. A vector, which comprises the nucleic acid moleculeaccording to claim 13; preferably, the vector is a cloning vector or anexpression vector.
 15. A host cell, which comprises the nucleic acidmolecule according to claim 13 or the vector according to claim
 14. 16.A method for preparing the antibody or antigen-binding fragment thereofaccording to any one of claims 1 to 12, which comprises culturing thehost cell according to claim 15 under a condition that allows theexpression of the antibody or antigen-binding fragment thereof, andrecovering the antibody or antigen-binding fragment thereof from thecultured host cell culture.
 17. A pharmaceutical composition, whichcomprises the antibody or antigen-binding fragment thereof according toany one of claims 1 to 12, and a pharmaceutically acceptable carrierand/or excipient.
 18. Use of the antibody or antigen-binding fragmentthereof according to any one of claims 1 to 12 or the pharmaceuticalcomposition according to claim 17 in the manufacture of a medicament forthe prevention and/or treatment of an HBV infection or HBVinfection-associated disease (for example, hepatitis B) in a subject(for example, a human), for neutralizing a virulence of HBV in vitro orin a subject (for example, a human), for reducing a serum level of HBVDNA and/or HBsAg in a subject (for example, a human), and/or foractivating a humoral immune response against HBV in a subject (forexample, a person with chronic HBV infection or a patient with chronichepatitis B).
 19. The antibody or antigen-binding fragment thereofaccording to any one of claims 1 to 12 or the pharmaceutical compositionaccording to claim 17, for use in the prevention and/or treatment of anHBV infection or HBV infection-associated disease (for example,hepatitis B) in a subject (for example, a human), for use inneutralizing a virulence of HBV in vitro or in a subject (for example, ahuman), for use in reducing a serum level of HBV DNA and/or HBsAg in asubject (for example, a human), and/or for use in activating a humoralimmune response against HBV in a subject (for example, a person withchronic HBV infection or a patient with chronic hepatitis B).
 20. Amethod, which is used for the prevention and/or treatment of an HBVinfection or HBV infection-associated disease (for example, hepatitis B)in a subject, for neutralizing a virulence of HBV in a subject (forexample, a human), for reducing a serum level of HBV DNA and/or HBsAg ina subject (for example, a human), and/or for activating a humoral immuneresponse against HBV in a subject (for example, a person with chronicHBV infection or a patient with chronic hepatitis B), the methodcomprises administering an effective amount of the antibody orantigen-binding fragment thereof according to any one of claims 1 to 12,or the pharmaceutical composition according to claim 17 to a subject inneed thereof.